Spatial mapping of nucleic acid sequence information

ABSTRACT

Presented are methods and compositions for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variations in tissues while preserving spatial information about the tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage Application of InternationalApplication No. PCT/US2016/043385, filed Jul. 21, 2016, which claims thebenefit of Provisional Application No. 62/197,389, filed on Jul. 27,2015, and claims the benefit of Provisional Application No. 62/218,742,filed Sep. 15, 2015, and claims the benefit of Provisional ApplicationNo. 62/250,329, filed Nov. 3, 2015, and claims the benefit ofProvisional Application No. 62/261,707, filed on Dec. 1, 2015, andclaims the benefit of Provisional Application No. 62/269,614, filed onDec. 18, 2015, all of which are herein incorporated by reference intheir entirety.

1. BACKGROUND

Existing techniques for the detection and analysis of nucleic acid(e.g., mRNA or genomic DNA) in a tissue sample typically provide spatialor localized information for one or limited number of genes at a time orprovide information for all of the genes in the sample without thedesired positional information. Recent interest has focused on thedevelopment of techniques that allow the characterization oftranscriptomes and/or genomic variations in tissues while preservingspatial information about the tissue. There is a need for methods ofcharacterizing nucleic acid in the context of a tissue sample.

2. SUMMARY

The present disclosure provides methods and compositions that facilitatethe characterization of transcriptomes and/or genomic variation intissues while preserving spatial information related to the origin oftarget nucleic acids in the tissue. For example, the methods disclosedherein can enable the identification of the location of a cell or a cellcluster in a tissue biopsy that carries an aberrant mutation. Themethods provided herein can therefore be useful for diagnostic purposes,e.g., for the diagnosis of cancer, and possibly aid in the selection oftargeted therapies.

The present disclosure provides a capture array for spatial detectionand analysis of nucleic acids in a tissue sample, comprising a capturesite comprising a pair of capture probes immobilized on a surface,wherein a first capture probe of the pair of capture probes comprises afirst primer binding region and a spatial address region, and wherein asecond capture probe of the pair of capture probes comprises a secondprimer binding region and a capture region.

The present disclosure also provides a method for spatial detection andanalysis of nucleic acids in a tissue sample that includes (a) providinga capture array, comprising a capture site comprising a pair of captureprobes immobilized on a surface, wherein a first capture probe of thepair of capture probes comprises a first primer binding region and aspatial address region, and wherein a second capture probe of the pairof capture probes comprises a second primer binding region and a captureregion.

The present disclosure also provides a method for spatial detection andanalysis of nucleic acids in a tissue sample that includes providing amagnetic nanoparticle comprising an immobilized capture probe comprisinga capture region.

The present disclosure also provides a method for spatial detection andanalysis of nucleic acids in a tissue sample that includes providing amagnetic nanoparticle comprising an immobilized capture probe comprisinga capture region.

The present disclosure also provides a capture array for spatialdetection and analysis of nucleic acids in a tissue sample, comprising acapture site comprising a capture probe comprising a spatial addressregion, and a transposon end (TE) region.

The present disclosure also provides a method for spatial detection andanalysis of nucleic acids in a tissue sample that includes providing acapture array comprising a capture site comprising a capture probecomprising a spatial address region and a transposon end (TE) region.

3. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a plan view of an exemplary embodiment of a capturearray for capture of nucleic acid in a tissue sample.

FIG. 1B illustrates a side view of one capture site of the capturearray, wherein the one capture site comprises at least one capture probefor capture of nucleic acid in a tissue sample.

FIG. 1C illustrates a side view of an exemplary embodiment of auniversal capture bead for capture of nucleic acid in a tissue sample.

FIG. 2 illustrates a flow diagram of an exemplary embodiment of a methodof spatial detection and analysis of nucleic acid in a tissue sample.

FIG. 3 illustrates a side view of the array of FIG. 1A and shows anexemplary embodiment of a process of capturing total mRNA in a tissuesample onto the array.

FIG. 4 illustrates a side view of the array of FIG. 1 and shows anexemplary embodiment of a process of capturing targeted mRNAs in atissue sample onto the array.

FIG. 5 illustrates a flow diagram of an exemplary embodiment of a methodof generating cDNA by in situ reverse transcription of RNA in a tissuesample for capture onto an array.

FIG. 6 illustrates an exemplary embodiment of the steps of a method ofFIG. 5.

FIG. 7 illustrates another exemplary embodiment of the steps of a methodof FIG. 5.

FIG. 8 illustrates a flow diagram of an exemplary embodiment of a methodof capturing DNA amplicons onto an array, wherein the DNA amplicons aregenerated by in situ amplification of target nucleic acid.

FIG. 9 illustrates an exemplary embodiment of the steps of a method ofFIG. 8.

FIG. 10 illustrates a flow diagram of an exemplary embodiment of amethod of capturing DNA amplicons onto an array, wherein the DNAamplicons are generated by in situ amplification of target nucleic acid.

FIG. 11 illustrates the steps of a method of FIG. 10.

FIG. 12 illustrates a flow diagram of an exemplary embodiment of amethod of capturing cDNA onto an array by single-strand ligation,wherein the cDNA is generated by in situ reverse transcription of targetRNA molecules.

FIGS. 13A and 13B illustrates the steps of a method of FIG. 12.

FIG. 14 illustrates a flow diagram of an exemplary embodiment of amethod of capturing DNA amplicons onto an array, wherein the DNAamplicons are generated by in situ amplification of target nucleic acid.

FIGS. 15A, 15B, and 15C illustrate the steps of a method of FIG. 14.

FIG. 16 illustrates a side view of a portion of an electrophoretictransfer system that is configured for spatial detection and analysis ofnucleic acid in a tissue sample.

FIG. 17 illustrates a side view of one capture site on a capture array(e.g., the capture array of FIG. 1A), wherein the one capture sitecomprises two separate sets of immobilized capture probes.

FIG. 18 illustrates a flow diagram of an embodiment of a method oftransferring nucleic acids from a tissue sample to a capture array forgeneration of a spatially addressed sequencing library, wherein thecapture array comprises capture sites that include separate pairs ofimmobilized capture probes, e.g., as shown in FIG. 17.

FIGS. 19A, 19B, 19C, and 19D illustrate the steps of the method of FIG.18.

FIG. 20 shows an exemplary embodiment of a process of capturing anucleic acid in a tissue sample for subsequent anchoring onto an array.

FIGS. 21A and 21B illustrate a grid array of a one-dimensional indexingscheme and a grid array of a two-dimensional indexing scheme,respectively, for spatial detection and analysis of nucleic acids in atissue sample.

FIG. 22 illustrates a flow diagram of an exemplary embodiment of amethod of using a combinatorial indexing system for generation of aspatially addressed cDNA sequencing library.

FIGS. 23A and 23B illustrate the steps of a method of FIG. 21.

FIG. 24 illustrates a flow diagram of an exemplary embodiment of analternative method of using a combinatorial indexing system forgeneration of a spatially addressed cDNA sequencing library.

FIGS. 25A, 25B, and 25C illustrate the steps of a method of FIG. 24.

FIG. 26A illustrates a plan view of an exemplary embodiment of an arrayfor delivery of reverse transcription (RT) primers to a tissue samplefor in situ synthesis of cDNA.

FIG. 26B illustrates a side view of a portion of one delivery site ofthe array of FIG. 26A, wherein the portion of the delivery sitecomprises at least one RT primer for synthesis of cDNA from mRNA in atissue sample.

FIG. 27A illustrates a plan view of an exemplary embodiment of a capturearray for the capture of cDNA synthesized in situ using the RT primersof FIG. 26B.

FIG. 27B illustrates a side view of a portion of one capture site of thecapture array of FIG. 27A, wherein the portion of the capture sitecomprises at least one capture probe for capture of cDNA synthesized.

FIG. 28 illustrates a flow diagram of an exemplary embodiment of amethod of generating a spatially addressed sequencing library, wherein afirst array is used for in situ synthesis of first strand cDNA and asecond array is used to capture the cDNA for subsequent librarygeneration.

FIGS. 29A and 29B illustrate the steps of a method of FIG. 28.

FIG. 30 illustrates a flow diagram of an exemplary embodiment of amethod of generating a spatially addressed cDNA library using releasablecapture probes.

FIGS. 31A and 31B illustrate exemplary schematic diagrams of a spatiallyaddressed capture probe that comprises a 5′ disulfide modification and aspatially addressed capture probe that comprises a 5′ photocleavablelinker, respectively.

FIGS. 32A, 32B, and 32C illustrate an exemplary embodiment of a processof reversibly anchoring the spatially addressed capture probe of FIG.31A onto the surface of a glass coverslip.

FIGS. 33A, 33B, and 33C illustrate an exemplary embodiment of a processof reversibly anchoring the spatially addressed capture probes of FIG.31B onto the surface of a glass coverslip.

FIGS. 34A and 34B illustrate the steps of the method of FIG. 30.

FIG. 35 illustrates a schematic diagram of an exemplary embodiment of acapture probe pair for capturing a genomic DNA region of interest.

FIG. 36 illustrates a flow diagram of an exemplary embodiment of amethod of generating a spatially addressed genomic amplicon libraryusing releasable capture probes.

FIG. 37 illustrates the steps of a method of FIG. 36.

FIG. 38 illustrates a flow diagram of an exemplary embodiment of amethod of generating a spatially addressed sequencing library usingmagnetic nanoparticles to capture nucleic acid from a tissue sample.

FIG. 39 illustrates the steps of a method of FIG. 38.

FIGS. 40A and 40B illustrate an example of a process of using a captureprobe to form a complementary nucleic acid in a tissue sample andsubsequently immobilizing the complementary nucleic acid to ananoparticle.

FIGS. 41A and 41B illustrate schematic diagrams of an example of aparticle-associated capture probe comprising a first partial spatialaddress region and a second array capture probe comprising a secondpartial address region, respectively, for spatial detection and analysisof nucleic acids in a tissue sample.

FIGS. 42A and 42B illustrate schematic diagrams of an example of aparticle-associated capture probe and a second array capture probecomprising a spatial address region, respectively, for spatial detectionand analysis of nucleic acids in a tissue sample.

FIGS. 43A and 43B illustrate schematic diagrams of an example of aparticle-associated capture probe and a second array capture probe,respectively, for spatial detection and analysis of nucleic acids from atissue sample.

FIG. 44 illustrates a perspective view of a magnetic-based transfersystem that is configured for spatial detection and analysis of nucleicacid in a tissue sample.

FIG. 45 illustrates a side view of one capture site on the capture arrayof FIG. 44, wherein the one capture site includes a plurality of captureprobes.

FIG. 46 illustrates a flow diagram of an example of a method oftransferring cDNA from a tissue sample to a capture array for generationof a spatially addressed sequencing library using the magnetic-basedtransfer system of FIG. 44 and FIG. 45.

FIGS. 47A, 47B, and 47C show pictorially the steps of the method of FIG.46.

FIG. 48 illustrates a flow diagram of an example of a method oftransferring RNA from a tissue sample to a capture array for generationof a spatially addressed sequencing library using the magnetic-basedtransfer system of FIG. 44.

FIGS. 49A, 49B, and 49C show pictorially the steps of the method of FIG.48.

FIG. 50 illustrates a flow diagram of an example of a method ofprofiling genomic DNA in a tissue sample.

FIG. 51 illustrates a diagram of a spatially addressed PCR primer forpre-amplification and spatial indexing of whole genomic DNA.

FIGS. 52A and 52B show pictorially the steps of the method of FIG. 50.

FIG. 53 illustrates a perspective view of an example of a microwellreactor overlay.

FIG. 54 illustrates a perspective view of a single microwell of FIG. 53.

FIGS. 55A and 55B illustrate an example of a process of fabricating themicrowell substrate of FIG. 53.

FIG. 56 illustrates a side view of an example of a microwell structurefor capture and spatial compartmentalization of nucleic acids from atissue sample.

FIG. 57 illustrates a flow diagram of an example of a method ofcapturing nucleic acids from a tissue section using the microwellstructure of FIG. 56 for preparation of a sequencing library.

FIG. 58A illustrates a side view of an example of a pin system fortissue excision and preparation of a spatially addressed nucleic acidlibrary.

FIG. 58B illustrates examples of different excision surfaces for thepins on the pin structure of FIG. 58A.

FIG. 59 illustrates a flow diagram of an example of a method ofcapturing nucleic acids from a tissue section using the pin system ofFIG. 58 for preparation of a sequencing library.

FIG. 60 illustrates side views of the pin system of FIG. 58 and showspictorially the steps 5910 and 5915 of the method of FIG. 59.

FIG. 61 illustrates a flow diagram of another example of a method ofcapturing nucleic acids from a tissue section using the pin system ofFIG. 58 for preparation of a sequencing library.

FIG. 62 illustrates side views of the pin system of FIG. 58 and showspictorially the steps 6110 and 6115 of the method of FIG. 61.

FIG. 63 illustrates a prespective view of a capillary “microreactor”system for capture of nucleic acids from a tissue section forpreparation of a spatially addressed nucleic acid library

FIG. 64 illustrates a flow diagram of an example of a method ofcapturing nucleic acids from a tissue section using the capillarymicroreactor system of FIG. 63 for preparation of a sequencing library.

FIG. 65 illustrates a side view of a portion of a droplet actuator thatis configured for spatial detection and analysis of nucleic acids in atissue sample.

FIG. 66 illustrates a side view of the pore sheet of FIG. 65.

FIGS. 67A and 67B illustrate side views of the droplet actuator of FIG.65 and show a process of isolating nucleic acid in a tissue sample forspatial detection and analysis.

FIG. 68 illustrates an exemplary embodiments of a method of generating aspatially addressed genomic amplicon library using tagmentation of wholegenomic DNA.

FIGS. 69A, 69B, and 69C illustrate the steps of a method of FIG. 68.

FIG. 70 illustrates a plan view of a spatial address overlay.

FIG. 71 illustrates a plan view of a single spatial feature on thesubstrate of FIG. 70.

4. DETAILED DESCRIPTION

Described herein are a variety of methods and compositions that allowfor the characterization of analytes in tissues while preserving spatialinformation related to the origin of target analyte in the tissue. Invarious embodiments, an array includes a substrate on which a pluralityof capture probes are immobilized such that each capture probe occupiesa distinct position on the array. Each capture probe includes, amongother sequences and/or molecules, a unique positional nucleic acid tag(i.e., a spatial address or indexing sequence). Each spatial addresscorresponds to the position of the capture probe on the array. Theposition of the capture probe on the array may be correlated with aposition in the tissue sample.

Examples of analytes in a tissue sample include genomic DNA, methylatedDNA, specific methylated DNA sequences, messenger RNA (mRNA), polyAmRNA, fragmented mRNA, fragmented DNA, mitochondrial DNA, viral RNA,microRNA, in situ synthesized PCR products, RNA/DNA hybrids, lipid,carbohydrate, protein, glycoprotein, lipoprotein, phosphoprotein,specific phosphorylated or acetylated variant of a protein, or viralcoat proteins.

A nucleic acid tag encoding location (i.e., a spatial address orindexing sequence) can be coupled to a nucleic acid capture region orany other molecule that binds a target analyte. Examples of othermolecules that may be coupled to a nucleic acid tag include antibodies,antigen binding domains, proteins, peptides, receptors, haptens, etc.

Described herein are a variety of methods and compositions that allowfor the characterization of transcriptomes and/or genomic variation intissues while preserving spatial information related to the origin oftarget nucleic acids in the tissue. For example, the methods disclosedherein can enable the identification of the location of a cell or a cellcluster in a tissue biopsy that carries an aberrant mutation. Themethods provided herein can therefore be useful for diagnostic purposes,e.g., for the diagnosis of cancer, and possibly aid in the selection oftargeted therapies.

The present disclosure is based, in part, on the realization thatinformation related to the spatial origin of a nucleic acid in a tissuesample can be encoded in the nucleic acid in the process of preparingthe nucleic acid for sequencing. For example, nucleic acids from atissue sample can be tagged by probes including location-specificsequence information (a “spatial address”). Spatially addressed nucleicacid molecules from a tissue sample can then be sequenced in bulk. Thesequence-identical nucleic acid molecules originating from differentregions in a tissue sample can be distinguished based on their spatialaddress and can be mapped onto their regions of origin in the tissuesample.

The present disclosure is further based, in part, on the realizationthat distinguishing related nucleic acids based on their spatial originin a tissue sample can increase the sensitivity of detection of raremutations in a complex tissue. For example, it was found that spatialaddressing of nucleic acids could increase the sensitivity of detectionof single nucleotide variations (SNVs) in a tissue sample.

In some methods described herein probes for spatial tagging can include,e.g., combinations of spatial address regions and gene-specific captureregions. The spatially addressed and gene-specific probes can becontacted with the tissue sample as immobilized probes on a capturearray. Alternatively, the spatially addressed probes can be releasedfrom the capture array and interact with the nucleic acids in solutionin the tissue sample, e.g., in situ.

The present disclosure is further based, in part, on the realizationthat spatial tagging can be performed using probes that separate spatialaddress regions from gene-specific capture regions. The ability toseparate capture regions from spatial address regions in two or moreprobe can increase the flexibility of sequencing library designs and oflibrary preparation protocols.

The present disclosure is further based, in part, on the realizationthat the robustness and data quality of spatial transcriptomicsexperiments can be enhanced by facilitating the transfer of nucleicacids from a tissue sample onto a capture array, e.g., a capture arrayof spatially addressed capture probes. For example, electrophoretictransfer of nucleic acids can be used to improve transfer yields andtransfer kinetics of nucleic acids. High-yield nucleic acid transferfrom tissue samples onto capture arrays can facilitate the detection ofrare SNVs. Fast transfer kinetics can be used limit nucleic aciddiffusion during the transfer process and help increase the resolutionof spatial addressing. Other methods described herein involve the use ofintermediate nucleic acid substrates, such as particles (e.g.,electromagnetic nanoparticles), membranes (e.g., nylon membranes) ormicrowell plates to facilitate nucleic acid capture in the tissuesample, to facilitate nucleic acid transfer onto capture arrays, and tolimit diffusion and improve spatial resolution. Additional methods,involving, e.g., tagmentation of genomic DNA are described that can beused to efficiently add spatial addresses to nucleic acids, e.g., on thesurface of a capture array.

The present disclosure is further based on the realization that spatialaddressing of nucleic acids from a tissue sample can involvetwo-dimensional spatial addressing, e.g., to correlate the position of anucleic acid on a two-dimensional capture array with the position of thenucleic acid in a two-dimensional tissue section. Spatial addressing canbe performed also in additional dimensions. For example, spatial addresssequences can be added to nucleic acids to describe the relative spatialposition of a nucleic acid in a third or fourth dimension, e.g., bydescribing the position of a tissue section in a tissue biopsy, or theposition of a tissue biopsy in a subject's organ. Temporal addresssequences could be added to nucleic acids from a tissue sample to denotea timepoint in a timecourse experiment, e.g., inquiring into changes ofgene-expression in a cell in response to a physical or chemicalstimulus, such as a drug treatment during a clinical trial.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “a capture probe” includes a mixture of two or more captureprobes, and the like.

The term “about,” particularly in reference to a given quantity, ismeant to encompass deviations of plus or minus five percent.

As used herein, the terms “includes,” “including,” “includes,”“including,” “contains,” “containing,” and any variations thereof, areintended to cover a non-exclusive inclusion, such that a process,method, product-by-process, or composition of matter that includes,includes, or contains an element or list of elements does not includeonly those elements but can include other elements not expressly listedor inherent to such process, method, product-by-process, or compositionof matter.

As used herein, the terms “address,” “tag,” or “index,” when used inreference to a nucleotide sequence is intended to mean a uniquenucleotide sequence that is distinguishable from other indices as wellas from other nucleotide sequences within polynucleotides containedwithin a sample. A nucleotide “address,” “tag,” or “index” can be arandom or a specifically designed nucleotide sequence. An “address,”“tag,” or “index” can be of any desired sequence length so long as it isof sufficient length to be unique nucleotide sequence within a pluralityof indices in a population and/or within a plurality of polynucleotidesthat are being analyzed or interrogated. A nucleotide “address,” “tag,”or “index” of the disclosure is useful, for example, to be attached to atarget polynucleotide to tag or mark a particular species foridentifying all members of the tagged species within a population.Accordingly, an index is useful as a barcode where different members ofthe same molecular species can contain the same index and wheredifferent species within a population of different polynucleotides canhave different indices.

As used herein, a “spatial address,” “spatial tag” or “spatial index,”when used in reference to a nucleotide sequence, means an address, tagor index encoding spatial information related to the region or locationof origin of an addressed, tagged, or indexed nucleic acid in a tissuesample.

As used herein, the term “substrate” is intended to mean a solidsupport. The term includes any material that can serve as a solid orsemi-solid foundation for creation of features such as wells for thedeposition of biopolymers, including nucleic acids, polypeptide and/orother polymers. A substrate as provided herein is modified, for example,or can be modified to accommodate attachment of biopolymers by a varietyof methods well known to those skilled in the art. Exemplary types ofsubstrate materials include glass, modified glass, functionalized glass,inorganic glasses, microspheres, including inert and/or magneticparticles, plastics, polysaccharides, nylon, nitrocellulose, ceramics,resins, silica, silica-based materials, carbon, metals, an optical fiberor optical fiber bundles, a variety of polymers other than thoseexemplified above and multiwell microtiter plates. Specific types ofexemplary plastics include acrylics, polystyrene, copolymers of styreneand other materials, polypropylene, polyethylene, polybutylene,polyurethanes and Teflon™. Specific types of exemplary silica-basedmaterials include silicon and various forms of modified silicon.

Those skilled in the art will know or understand that the compositionand geometry of a substrate as provided herein can vary depending on theintended use and preferences of the user. Therefore, although planarsubstrates such as slides, chips or wafers are exemplified herein inreference to microarrays for illustration, given the teachings andguidance provided herein, those skilled in the art will understand thata wide variety of other substrates exemplified herein or well known inthe art also can be used in the methods and/or compositions herein.

In some embodiments, the solid support comprises one or more surfaces ofa flowcell. The term “flowcell” as used herein refers to a chambercomprising a solid surface across which one or more fluid reagents canbe flowed. Examples of flowcells and related fluidic systems anddetection platforms that can be readily used in the methods of thepresent disclosure are described, for example, in Bentley et al., Nature456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO 91/06678; WO07/123744; U.S. Pat. Nos. 7,329,492; 7,211,414; 7,315,019; 7,405,281,and US 2008/0108082, each of which is incorporated herein by reference.

In some embodiments, the solid support includes a patterned surface. A“patterned surface” refers to an arrangement of different regions in oron an exposed layer of a solid support. For example, one or more of theregions can be features where one or more amplification primers arepresent. The features can be separated by interstitial regions whereamplification primers are not present. In some embodiments, the patterncan be an x-y format of features that are in rows and columns. In someembodiments, the pattern can be a repeating arrangement of featuresand/or interstitial regions. In some embodiments, the pattern can be arandom arrangement of features and/or interstitial regions. Exemplarypatterned surfaces that can be used in the methods and compositions setforth herein are described in U.S. Ser. No. 13/661,524 or US Pat. App.Publ. No. 2012/0316086 A1, each of which is incorporated herein byreference.

As used herein, the term “interstitial region” refers to an area in asubstrate or on a surface that separates other areas of the substrate orsurface. For example, an interstitial region can separate one feature ofan array from another feature of the array. The two regions that areseparated from each other can be discrete, lacking contact with eachother. In another example, an interstitial region can separate a firstportion of a feature from a second portion of a feature. The separationprovided by an interstitial region can be partial or full separation.Interstitial regions will typically have a surface material that differsfrom the surface material of the features on the surface. For example,features of an array can have an amount or concentration of captureagents or primers that exceeds the amount or concentration present atthe interstitial regions. In some embodiments the capture agents orprimers may not be present at the interstitial regions.

In some embodiments, the solid support includes an array of wells ordepressions in a surface. This may be fabricated as is generally knownin the art using a variety of techniques, including, but not limited to,photolithography, stamping techniques, molding techniques andmicroetching techniques. As will be appreciated by those in the art, thetechnique used will depend on the composition and shape of the arraysubstrate.

The features in a patterned surface can be wells in an array of wells(e.g., microwells or nanowells) on glass, silicon, plastic or othersuitable solid supports with patterned, covalently-linked gel such aspoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM, see,for example, U.S. Prov. Pat. App. Ser. No. 61/753,833, which isincorporated herein by reference). The process creates gel pads used forsequencing that can be stable over sequencing runs with a large numberof cycles. The covalent linking of the polymer to the wells is helpfulfor maintaining the gel in the structured features throughout thelifetime of the structured substrate during a variety of uses. Howeverin many embodiments, the gel need not be covalently linked to the wells.For example, in some conditions silane free acrylamide (SFA, see, forexample, U.S. Pat. App. Pub. No. 2011/0059865 A1, which is incorporatedherein by reference) which is not covalently attached to any part of thestructured substrate, can be used as the gel material.

In particular embodiments, a structured substrate can be made bypatterning a solid support material with wells (e.g. microwells ornanowells), coating the patterned support with a gel material (e.g.,PAZAM, SFA or chemically modified variants thereof, such as theazidolyzed version of SFA (azido-SFA)) and polishing the gel coatedsupport, for example via chemical or mechanical polishing, therebyretaining gel in the wells but removing or inactivating substantiallyall of the gel from the interstitial regions on the surface of thestructured substrate between the wells. Primer nucleic acids can beattached to gel material. A solution of target nucleic acids (e.g., afragmented human genome) can then be contacted with the polishedsubstrate such that individual target nucleic acids will seed individualwells via interactions with primers attached to the gel material;however, the target nucleic acids will not occupy the interstitialregions due to absence or inactivity of the gel material. Amplificationof the target nucleic acids will be confined to the wells since absenceor inactivity of gel in the interstitial regions prevents outwardmigration of the growing nucleic acid colony. The process isconveniently manufacturable, being scalable and utilizing conventionalmicro- or nano-fabrication methods.

A patterned substrate can include, for example, wells etched into aslide or chip. The pattern of the etchings and geometry of the wells cantake on a variety of different shapes and sizes so long as such featuresare physically or functionally separable from each other. Particularlyuseful substrates having such structural features are patternedsubstrates that can select the size of solid support particles such asmicrospheres. An exemplary patterned substrate having thesecharacteristics is the etched substrate used in connection withBeadArray technology (Illumina, Inc., San Diego, Calif.). Furtherexamples, are described in U.S. Pat. No. 6,770,441, which isincorporated herein by reference.

As used herein, the term “immobilized” when used in reference to anucleic acid is intended to mean direct or indirect attachment to asolid support via covalent or non-covalent bond(s). In certainembodiments, covalent attachment can be used, but all that is requiredis that the nucleic acids remain stationary or attached to a supportunder conditions in which it is intended to use the support, forexample, in applications requiring nucleic acid amplification and/orsequencing. Oligonucleotides to be used as capture primers oramplification primers can be immobilized such that a 3′-end is availablefor enzymatic extension and at least a portion of the sequence iscapable of hybridizing to a complementary sequence. Immobilization canoccur via hybridization to a surface attached oligonucleotide, in whichcase the immobilized oligonucleotide or polynucleotide can be in the3′-5′ orientation. Alternatively, immobilization can occur by meansother than base-pairing hybridization, such as the covalent attachmentset forth above.

Certain embodiments may make use of solid supports comprised of an inertsubstrate or matrix (e.g. glass slides, polymer beads etc.) which hasbeen functionalized, for example by application of a layer or coating ofan intermediate material comprising reactive groups which permitcovalent attachment to biomolecules, such as polynucleotides. Examplesof such supports include, but are not limited to, polyacrylamidehydrogels supported on an inert substrate such as glass, particularlypolyacrylamide hydrogels as described in WO 2005/065814 and US2008/0280773, the contents of which are incorporated herein in theirentirety by reference. In such embodiments, the biomolecules (e.g.polynucleotides) may be directly covalently attached to the intermediatematerial (e.g. the hydrogel) but the intermediate material may itself benon-covalently attached to the substrate or matrix (e.g. the glasssubstrate). The term “covalent attachment to a solid support” is to beinterpreted accordingly as encompassing this type of arrangement.

Exemplary covalent linkages include, for example, those that result fromthe use of click chemistry techniques. Exemplary non-covalent linkagesinclude, but are not limited to, non-specific interactions (e.g.hydrogen bonding, ionic bonding, van der Waals interactions etc.) orspecific interactions (e.g. affinity interactions, receptor-ligandinteractions, antibody-epitope interactions, avidin-biotin interactions,streptavidin-biotin interactions, lectin-carbohydrate interactions,etc.). Exemplary linkages are set forth in U.S. Pat. Nos. 6,737,236;7,259,258; 7,375,234 and 7,427,678; and US Pat. Pub. No. 2011/0059865A1, each of which is incorporated herein by reference.

As used herein, the term “array” refers to a population of sites thatcan be differentiated from each other according to relative location.Different molecules that are at different sites of an array can bedifferentiated from each other according to the locations of the sitesin the array. An individual site of an array can include one or moremolecules of a particular type. For example, a site can include a singletarget nucleic acid molecule having a particular sequence or a site caninclude several nucleic acid molecules having the same sequence (and/orcomplementary sequence, thereof). The sites of an array can be differentfeatures located on the same substrate. Exemplary features includewithout limitation, wells in a substrate, beads (or other particles) inor on a substrate, projections from a substrate, ridges on a substrateor channels in a substrate. The sites of an array can be separatesubstrates each bearing a different molecule. Different moleculesattached to separate substrates can be identified according to thelocations of the substrates on a surface to which the substrates areassociated or according to the locations of the substrates in a liquidor gel. Exemplary arrays in which separate substrates are located on asurface include, without limitation, those having beads in wells.

As used herein, the term “plurality” is intended to mean a population oftwo or more different members. Pluralities can range in size from small,medium, large, to very large. The size of small plurality can range, forexample, from a few members to tens of members. Medium sized pluralitiescan range, for example, from tens of members to about 100 members orhundreds of members. Large pluralities can range, for example, fromabout hundreds of members to about 1000 members, to thousands of membersand up to tens of thousands of members. Very large pluralities canrange, for example, from tens of thousands of members to about hundredsof thousands, a million, millions, tens of millions and up to or greaterthan hundreds of millions of members. Therefore, a plurality can rangein size from two to well over one hundred million members as well as allsizes, as measured by the number of members, in between and greater thanthe above exemplary ranges. An exemplary number of features within amicroarray includes a plurality of about 500,000 or more discretefeatures within 1.28 cm′. Exemplary nucleic acid pluralities include,for example, populations of about 1×10⁵, 5×10⁵ and 1×10⁶ or moredifferent nucleic acid species. Accordingly, the definition of the termis intended to include all integer values greater than two. An upperlimit of a plurality can be set, for example, by the theoreticaldiversity of nucleotide sequences in a nucleic acid sample.

As used herein, the term “nucleic acid” is intended to be consistentwith its use in the art and includes naturally occurring nucleic acidsor functional analogs thereof. Particularly useful functional analogsare capable of hybridizing to a nucleic acid in a sequence specificfashion or capable of being used as a template for replication of aparticular nucleotide sequence. Naturally occurring nucleic acidsgenerally have a backbone containing phosphodiester bonds. An analogstructure can have an alternate backbone linkage including any of avariety of those known in the art. Naturally occurring nucleic acidsgenerally have a deoxyribose sugar (e.g. found in deoxyribonucleic acid(DNA)) or a ribose sugar (e.g. found in ribonucleic acid (RNA)). Anucleic acid can contain any of a variety of analogs of these sugarmoieties that are known in the art. A nucleic acid can include native ornon-native bases. In this regard, a native deoxyribonucleic acid canhave one or more bases selected from the group consisting of adenine,thymine, cytosine or guanine and a ribonucleic acid can have one or morebases selected from the group consisting of uracil, adenine, cytosine orguanine Useful non-native bases that can be included in a nucleic acidare known in the art. The term “target,” when used in reference to anucleic acid, is intended as a semantic identifier for the nucleic acidin the context of a method or composition set forth herein and does notnecessarily limit the structure or function of the nucleic acid beyondwhat is otherwise explicitly indicated. Particular forms of nucleicacids may include all types of nucleic acids found in an organism aswell as synthetic nucleic acids such as polynucleotides produced bychemical synthesis. Particular examples of nucleic acids that areapplicable for analysis through incorporation into microarrays producedby methods as provided herein include genomic DNA (gDNA), expressedsequence tags (ESTs), DNA copied messenger RNA (cDNA), RNA copiedmessenger RNA (cRNA), mitochondrial DNA or genome, RNA, messenger RNA(mRNA) and/or other populations of RNA. Fragments and/or portions ofthese exemplary nucleic acids also are included within the meaning ofthe term as it is used herein.

As used herein, the term “double-stranded,” when used in reference to anucleic acid molecule, means that substantially all of the nucleotidesin the nucleic acid molecule are hydrogen bonded to a complementarynucleotide. A partially double stranded nucleic acid can have at least10%, 25%, 50%, 60%, 70%, 80%, 90% or 95% of its nucleotides hydrogenbonded to a complementary nucleotide.

As used herein, the term “single-stranded,” when used in reference to anucleic acid molecule, means that essentially none of the nucleotides inthe nucleic acid molecule are hydrogen bonded to a complementarynucleotide.

As used herein, the term “capture primers” is intended to mean anoligonucleotide having a nucleotide sequence that is capable ofspecifically annealing to a single stranded polynucleotide sequence tobe analyzed or subjected to a nucleic acid interrogation underconditions encountered in a primer annealing step of, for example, anamplification or sequencing reaction. The terms “nucleic acid,”“polynucleotide” and “oligonucleotide” are used interchangeably herein.The different terms are not intended to denote any particular differencein size, sequence, or other property unless specifically indicatedotherwise. For clarity of description the terms can be used todistinguish one species of nucleic acid from another when describing aparticular method or composition that includes several nucleic acidspecies.

As used herein, the term “gene-specific” or “target specific” when usedin reference to a capture probe or other nucleic acid is intended tomean a capture probe or other nucleic acid that includes a nucleotidesequence specific to a targeted nucleic acid, e.g., a nucleic acid froma tissue sample, namely a sequence of nucleotides capable of selectivelyannealing to an identifying region of a targeted nucleic acid.Gene-specific capture probes can have a single species ofoligonucleotide, or can include two or more species with differentsequences. Thus, the gene-specific capture probes can be two or moresequences, including 3, 4, 5, 6, 7, 8, 9 or 10 or more differentsequences. The gene-specific capture probes can comprise a gene-specificcapture primer sequence and a universal capture probe sequence. Othersequences such as sequencing primer sequences and the like also can beincluded in a gene-specific capture primer.

In comparison, the term “universal” when used in reference to a captureprobe or other nucleic acid is intended to mean a capture probe ornucleic acid having a common nucleotide sequence among a plurality ofcapture probes. A common sequence can be, for example, a sequencecomplementary to the same adapter sequence. Universal capture probes areapplicable for interrogating a plurality of different polynucleotideswithout necessarily distinguishing the different species whereasgene-specific capture primers are applicable for distinguishing thedifferent species.

In various embodiments, the capture elements (e.g., capture primers orcapture probes or other nucleic acid sequences) can be spaced to A)spatially resolve nucleic acids within the geometry of a single cell,i.e., multiple capture sites per cell; B) spatially resolve nucleicacids at about the single cell level, i.e., about 1 capture site percell. Additionally, capture elements may be spaced as in A or B above,and be: I) spaced to sample nucleic acids from a sample at regularintervals, e.g., spaced in a grid or pattern such that about every otheror every 5^(th) or every 10^(th) cell is sampled, or about every otheror every 5^(th) or every 10 gropu of 2, 3, 4, 5, 6, 7, 8, 9, 10 or morecells is sampled; II) spaced to capture samples from substantially allavailable cells in one or more regions of a sample, or III) spaced tocapture samples from substantially all available cells in the sample.

As used herein, the term “amplicon,” when used in reference to a nucleicacid, means the product of copying the nucleic acid, wherein the producthas a nucleotide sequence that is the same as or complementary to atleast a portion of the nucleotide sequence of the nucleic acid. Anamplicon can be produced by any of a variety of amplification methodsthat use the nucleic acid, or an amplicon thereof, as a templateincluding, for example, polymerase extension, polymerase chain reaction(PCR), rolling circle amplification (RCA), ligation extension, orligation chain reaction. An amplicon can be a nucleic acid moleculehaving a single copy of a particular nucleotide sequence (e.g. a PCRproduct) or multiple copies of the nucleotide sequence (e.g. aconcatameric product of RCA). A first amplicon of a target nucleic acidcan be a complementary copy. Subsequent amplicons are copies that arecreated, after generation of the first amplicon, from the target nucleicacid or from the first amplicon. A subsequent amplicon can have asequence that is substantially complementary to the target nucleic acidor substantially identical to the target nucleic acid.

The number of template copies or amplicons that can be produced can bemodulated by appropriate modification of the amplification reactionincluding, for example, varying the number of amplification cycles run,using polymerases of varying processivity in the amplification reactionand/or varying the length of time that the amplification reaction isrun, as well as modification of other conditions known in the art toinfluence amplification yield. The number of copies of a nucleic acidtemplate can be at least 1, 10, 100, 200, 500, 1000, 2000, 3000, 4000,5000, 6000, 7000, 8000, 9000 and 10,000 copies, and can be varieddepending on the particular application.

As used herein, the term “each,” when used in reference to a collectionof items, is intended to identify an individual item in the collectionbut does not necessarily refer to every item in the collection unlessthe context clearly dictates otherwise.

Provided herein are arrays for and methods of spatial detection andanalysis (e.g., mutational analysis or single nucleotide variation (SNV)detection) of nucleic acid in a tissue sample. The arrays describedherein can comprise a substrate on which a plurality of capture probesare immobilized such that each capture probe occupies a distinctposition on the array. Some or all of the plurality of capture probescan comprise a unique positional tag (i.e., a spatial address orindexing sequence). A spatial address can describe the position of thecapture probe on the array. The position of the capture probe on thearray can be correlated with a position in the tissue sample.

As used herein, the term “tissue sample” refers to a piece of tissuethat has been obtained from a subject, fixed, sectioned, and mounted ona planar surface, e.g., a microscope slide. The tissue sample can be aformalin-fixed paraffin-embedded (FFPE) tissue sample or a fresh tissuesample or a frozen tissue sample, etc. The methods disclosed herein maybe performed before or after staining the tissue sample. For example,following hematoxylin and eosin staining, a tissue sample may bespatially analyzed in accordance with the methods as provided herein. Amethod may include analyzing the histology of the sample (e.g., usinghematoxylin and esoins staining) and then spatially analyzing thetissue.

As used herein, the term “formalin-fixed paraffin embedded (FFPE) tissuesection” refers to a piece of tissue, e.g., a biopsy that has beenobtained from a subject, fixed in formaldehyde (e.g., 3%-5% formaldehydein phosphate buffered saline) or Bouin solution, embedded in wax, cutinto thin sections, and then mounted on a planar surface, e.g., amicroscope slide.

In some embodiments, nucleic acids in a tissue sample are transferred toand captured onto an array. For example, a tissue section is placed incontact with an array and nucleic acid is captured onto the array andtagged with a spatial address. The spatially-tagged DNA molecules arereleased from the array and analyzed, for example, by high throughputnext generation sequencing (NGS), such as sequencing-by-synthesis (SBS).In some embodiments, a nucleic acid in a tissue section (e.g., aformalin-fixed paraffin-embedded (FFPE) tissue section) is transferredto an array and captured onto the array by hybridization to a captureprobe. In some embodiments, a capture probe can be a universal captureprobe hybridizing, e.g., to an adaptor region in a nucleic acidsequencing library, or to the poly-A tail of an mRNA. In someembodiments, the capture probe can be a gene-specific capture probehybridizing, e.g., to a specifically targeted mRNA or cDNA in a sample,such as a TruSeq™ Custom Amplicon (TSCA) oligonucleotide probe(Illumina, Inc.). A capture probe can be a plurality of capture probes,e.g., a plurality of the same or of different capture probes.

In some embodiments, a nucleic acid in a tissue section (e.g., an FFPEsection) is transferred to an array and captured onto the array bysingle-strand ligation to a universal adaptor oligonucleotide. Forexample, universal adaptor oligonucleotides that include spatialaddresses can be immobilized on a bead array. Single-stranded nucleicacid targets can be ligated to the adaptors for capture. The nucleicacid can comprise cDNA or genomic DNA amplicons. The universal adaptorscan be used to capture gene-specific cDNA or DNA amplicons. Theorientation of universal adaptors on the array (e.g., bead array) can becontrolled to capture both 3′ and 5′ regions of target nucleic acids.

In some embodiments, a capture array (i.e., an array of capture sites)can be integrated with an electrophoretic system to facilitate thetransfer of nucleic acid molecules from a tissue section onto a capturesite on the array. Electrophoretic transfer of nucleic acids canmaintain spatial resolution about the tissue context by limiting thediffusion of nucleic acid molecules away from their location of originduring transfer and by thereby reducing loss of nucleic acids betweencapture sites.

In some embodiments, a combinatorial indexing (addressing) system isused to provide spatial information for analysis of nucleic acids in atissue sample. The combinatorial indexing system can involve the use oftwo or more spatial address sequences (e.g., two, three, four, five ormore spatial address sequences).

In some embodiments, two spatial address sequences are incorporated intoa nucleic acid during preparation of a sequencing library. A firstspatial address can be used to define a certain position (i.e., capturesite) in the X dimension on a capture array and a second spatial addresssequence can be used define a position (i.e., a capture site) in the Ydimension on the capture array. During library sequencing, both X and Yspatial address sequences can be determined and the sequence informationcan be analyzed to define the specific position on the capture array.

In some embodiments, three spatial address sequences are incorporatedinto a nucleic acid during preparation of a sequencing library. A firstspatial address can be used to define a certain position (i.e., capturesite) in the X dimension on a capture array, a second spatial addresssequence can be used define a position (i.e., a capture site) in the Ydimension on the capture array, and a third spatial address sequence canbe used to define a position of a two-dimensional sample section (e.g.,the position of a slice of a tissue sample) in a sample (e.g., a tissuebiopsy) to provide positional spatial information in the third dimension(Z dimension) of a sample. During library sequencing, X, Y, and Zspatial address sequences can be determined and the sequence informationcan be analyzed to define the specific position on the capture array.

In some embodiments, a temporal address sequence (T) is optionallyincorporated into a nucleic acid during preparation of a sequencinglibrary. In some embodiments, the temporal address sequence can becombined with two or three spatial address sequences. The temporaladdress sequence can, for example, be used in the context of atime-course experiment for determining time-dependent changes ingene-expression in a tissue sample. Time-dependent changes ingene-expression can occur in a tissue sample, for example, in responseto a chemical, biological or physical stimulus (e.g., a toxin, a drug,or heat). Nucleic acid samples obtained at different timepoints fromcomparable tissue samples (e.g., proximal slices of a tissue sample) canbe pooled and sequenced in bulk. An optional first spatial address canbe used to define a certain position (i.e., capture site) in the Xdimension on a capture array, a second optional spatial address sequencecan be used to define a position (i.e., a capture site) in the Ydimension on the capture array, and a third optional spatial addresssequence can be used to define a position of a two-dimensional samplesection (e.g., the position of a slice of a tissue sample) in a sample(e.g., a tissue biopsy) to provide positional spatial information in thethird dimension (Z dimension) of the sample. During library sequencing,T, X, Y, and Z address sequences are determined and the sequenceinformation is analyzed to define the specific X, Y (and optionally Z)position on the capture array for each timepoint (T).

The address sequences X, Y, and, optionally, Z and/or T, can beconsecutive nucleic acid sequences or the address sequences can beseparated by one or more nucleic acids (e.g., 2 or more, 3 or more, 10or more, 30 or more, 100 or more, 300 or more, or 1,000 or more). Insome embodiments, the X, Y, and optionally Z and/or T address sequencescan each individually and independently be combinatorial nucleic acidsequences.

In some embodiments, the length of the address sequences (e.g., X, Y, Z,or T) can each individually and independently be 100 nucleic acids orless, 90 nucleic acids or less, 80 nucleic acids or less, 70 nucleicacids or less, 60 nucleic acids or less, 50 nucleic acids or less, 40nucleic acids or less, 30 nucleic acids or less, 20 nucleic acids orless, 15 nucleic acids or less, 10 nucleic acids or less, 8 nucleicacids or less, 6 nucleic acids or less, or 4 nucleic acids or less. Thelength of two or more address sequences in a nucleic acid can be thesame or different. For example, if the length of address sequence X is10 nucleic acids, the length of address sequence Y can be, e.g., 8nucleic acids, 10 nucleic acids, or 12 nucleic acids.

Address sequences, e.g., spatial address sequences such as X or Y, canbe either partially or fully degenerate sequences.

In some embodiments, spatially addressed capture probes on an array canbe released from the array onto a tissue section for generation of aspatially addressed sequencing library. In some embodiments, a captureprobe comprises a random primer sequence for in situ synthesis ofspatially-tagged cDNA from RNA in the tissue section. In someembodiments, a capture probe is a TruSeq™ Custom Amplicon (TSCA)oligonucleotide probe (Illumina, Inc.) for capturing and spatiallytagging genomic DNA in the tissue section. The spatially-tagged nucleicacid molecules (e.g., cDNA or genomic DNA) are recovered from the tissuesection and processed in single tube reactions to generate aspatially-tagged amplicon library.

In some embodiments, magnetic nanoparticles can be used to capturenucleic acid (e.g., in situ synthesized cDNA) in a tissue sample forgeneration of a spatially addressed library.

In some embodiments, spatial detection and analysis of nucleic acid in atissue sample can be performed on a droplet actuator.

4.1 Generation of Arrays

In one aspect, provided herein are capture arrays comprising spatiallyencoded capture probes. The spatially encoded capture probes on thecapture probes can be immobilized, e.g., on a planar glass substrate, oron a plurality of beads.

FIG. 1A illustrates a plan view of an exemplary embodiment of a capturearray 100 for capture of nucleic acid in a tissue sample. Capture array100 comprises an arrangement (e.g., rows and columns) of capture sites105 on a solid support 110. In some embodiments, solid support 110 is aplanar glass substrate. In some embodiments, solid support 110 is a bead(e.g., see FIG. 1C). At least one capture probe 115 is immobilized ateach of the capture sites 105 of capture array 100, as shown in FIG. 1B.Namely, FIG. 1B illustrates a side view of one capture site 105 ofcapture array 100, wherein the one capture site 105 comprises at leastone capture probe 115 for capture of nucleic acid in a tissue sample.FIG. 1B shows a capture probe 115 immobilized on the surface of solidsupport 110. In this embodiment, a single capture probe 115 is shown,but any number of capture probes 115 can be immobilized on solid support110 at each capture site 105. Capture probe 115 may optionally comprisea cleavable sequence in a cleavable region 120, an SBS primer sequencein a SBS primer binding site 125, a spatial address sequence in aspatial address region 130, and a capture sequence in a capture region135. Cleavable region 120 can be used to release captured nucleic acidfrom capture array 100 such that spatial address region 130 is includedin the released nucleic acid and the nucleic acid is “tagged.” SBSprimer region 125 can comprise an SBS primer sequence (e.g., SBS12 orSBS3) that can be used in a sequencing-by-synthesis (SBS) process.Alternatively, SBS primer sequence 125 or some portion thereof may beadded subsequently, e.g., by ligation or by PCR synthesis. SBS primerregion 125 can also be used in an amplification reaction to generate asequencing library as described in more detail with reference to FIG. 12and FIG. 13. Spatial address region 130 corresponds to the position ofcapture probe 115 in capture array 100. Each capture probe 115 at acapture site 105 comprises a unique spatial address region 130. Theposition of capture probe 115 in capture array 100 can be correlatedwith a position in the tissue sample.

Capture region 135 can be, for example, a universal (general) captureregion. In some embodiments, capture region 135 comprises a poly-Toligonucleotide that can be used to capture total mRNA in a tissuesample as described in more detail with reference to FIG. 3. In someembodiments, capture region 135 is a universal capture region that canbe used to capture cDNA synthesized by in situ reverse transcription ofRNA as describe in more detail with reference to FIG. 6. In someembodiments, capture region 135 is a universal capture region that canbe used to capture genomic DNA amplicons as described in more detailwith reference to FIG. 9 and FIG. 10.

In some embodiments, capture region 135 is a gene-specific ortarget-specific capture region that can be used to capture a specificnucleic acid in a tissue sample. Each capture probe 115 on capture array100 can comprise one or more unique gene-specific capture region 135.U.S. Patent Pub. No. 2015148239, filed on Sep. 22, 2014 by Peter et al.,and incorporated herein by reference, describes cleavable PCR primers inwhich each probe includes multiple cleavable primers, and which may beemployed in this and other embodiments described herein. Differentcapture probes 115 on capture array 110 can have the same gene-specificcapture region or they can be different gene-specific capture regions.In some embodiments, the nucleic acid in the tissue sample is agene-specific mRNA as described in more detail with reference to FIG. 4.In some embodiments, the nucleic acid in the tissue sample is agene-specific cDNA synthesized by in situ reverse transcription of RNAas described in more detail with reference to FIG. 7. In someembodiments (not shown), capture region 135 is a gene-specific regionthat can be used to capture genomic DNA amplicons.

The probes may be contacted with tissue by placing the tissue directlyon the surface comprising the probes; placing the tissue on a substance,such as a filter or a gel or a thin buffer layer, separating the tissuefrom the probes such that the target nucleic acids may diffuse from thetissue, through the substance to the probes; placing the tissue on asubstance such as a filter or a gel or a thin buffer layer separatingthe tissue from the probes such that the probes may diffuse from thesurface comprising the probes, through the substance to the targets;extracting the targets from the tissue onto an intermediate substrate(e.g., a gel, filter, solid substrate, or combinations of theforegoing), which is then placed on the surface supporting the probes;and combinations of the foregoing. In each case, the technique isselected to substantially maintain information encoding the spatialorientation of the targets in the sample.

FIG. 1C illustrates a side view of an embodiment of a universal capturebead 150 for capture of nucleic acid in a tissue sample. In someembodiments, there is a universal capture bead 150 at each of thecapture sites 105 of capture array 100. For example, universal capturebeads 150 can be deposited into wells on solid support 110 (e.g., aglass substrate). Universal capture bead 150 comprises a bead 155. Auniversal adaptor oligonucleotide 160 is immobilized on the surface ofbead 155. Universal adaptor oligonucleotide 160 is essentially the sameas capture probe 115 of FIG. 1B except that capture region 135 isomitted, i.e., universal adaptor oligonucleotide 160 comprises onlycleavable region 120, SBS primer region 125, and spatial address region130. SBS primer region 125 can comprise, for example, an SBS12 sequenceor an SBS3 sequence. In this embodiment, a single universal adaptoroligonucleotide 160 is shown, but any number of universal adaptoroligonucleotides 160 can be immobilized on bead 155. Universal capturebead 150 can be used to capture nucleic acid in a tissue sample bysingle-strand ligation of target nucleic acid (e.g., cDNA or genomic DNAamplicons) to universal adaptor oligonucleotide 160 as described in moredetail with reference to FIGS. 12 through 15.

A gene-specific capture array, such as a bead array with a plurality ofgene-specific capture probes on each bead, can be produced using aligation-based approach. For example, a bead array can be designed tohave 1 million spatial addresses on a bead. The array can be designed tocapture nucleic acid from 1,000 genes. To capture nucleic acid from1,000 genes on a bead designed to have 1 million spatial addresses wouldrequire 1 billion capture probes (i.e., 1,000 genes×1 million spatialaddresses=1 billion capture oligonucleotides). To avoid the synthesis of1 billion capture probes, a pool of oligonucleotides representinggene-specific capture regions (e.g., capture region 135) can be ligatedonto spatially addressed capture probes comprising cleavage region 120,SBS primer region 125, and spatial address region 130 (e.g.,oligonucleotides representing 1,000 gene-specific capture regions+1million spatial addressregions=1.1 million capture probes). In someembodiments, the pool of gene-specific capture regions is ligated to thespatially addressed capture probes using an enzymatic ligation approach.In some embodiments, the pool of gene-specific capture regions isligated to the spatially addressed capture probes using a chemicalligation approach.

A ligation-based approach can also be used to produce a plurality ofspatial addresses for a bead array. The current approach to produce aspatially-addressed bead array requires synthesis of eacholigonucleotide independently for each distinct spatial address (e.g., 1million spatial addresses requires synthesis of 1 millionoligonucleotides). To avoid synthesizing 1 million oligonucleotides, acombinatorial approach can be used. For example, three distinct subsetsof oligonucleotides with unique sequences (e.g., subset A with 100unique sequence, subset B with 100 unique sequences, and subset C with100 unique sequences) are synthesized and used in a combinatorialligation reaction, e.g., 100 subset A×100 subset B×100 subset C=1million oligonucleotides with distinct spatial addresses. Thecombinatorial approach requires the synthesis of only 300 differentoligonucleotides.

In some embodiments, a hybridization and extension approach can be usedto produce spatially addressed gene-specific capture probes. Forexample, a set “X” of 1,000 oligonucleotides with unique spatialaddresses is synthesized. A second set “Y” of 1000 oligonucleotides thatindividually captures a unique gene and can hybridize to set “X”oligonucleotides is synthesized. Each individual oligo of set “Y”oligonucleotides are hybridized to set “X” oligonucleotides and anextension reaction is performed. Using this approach, synthesis of 2,000oligonucleotides is required to generate 1 million different captureprobes (1,000 unique spatial address sequence individually paired with1,000 different gene-specific capture sequence). Using generaloligonucleotide synthesis, the production of 1,000 gene-specific captureprobes with each individually having 1,000 different spatial addresswould require the synthesis of 1 million oligonucleotide (1,000genes×1,000 address).

The beads comprising the probes may be contacted with tissue by placingthe tissue directly on the surface comprising the beads; placing thetissue on a substance, such as a filter or a gel or a thin buffer layer,separating the tissue from the beads such that the target nucleic acidsmay diffuse from the tissue, through the substance to the probes;placing the tissue on a substance such as a filter or a gel or a thinbuffer layer separating the tissue from the probes such that the probesmay diffuse from the beads, through the substance to the targets;extracting the targets from the tissue onto an intermediate substrate(e.g., a gel, filter, solid substrate, or combinations of theforegoing), which is then placed on the surface supporting the beads;depositing the beads directly into the tissue; and combinations of theforegoing. In each case, the technique is selected to substantiallymaintain information encoding the spatial orientation of the targets inthe sample.

4.2 Spatial Detection and Analysis of Nucleic Acid in a Tissue Sample

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a sample.

FIG. 2 illustrates a flow diagram of an embodiment of a method 200 ofspatial detection and analysis of a nucleic acid in a tissue sample.Method 200 can include, but is not limited to, some or all of thefollowing steps.

At a step 210, a tissue sample is prepared for analysis. In someembodiments, the tissue sample is a FFPE tissue sample that is sectionedonto a slide. Other examples include fresh tissue, frozen tissue, etc.

At a step 215, in situ biochemistry is performed on the tissue sectionto facilitate subsequent manipulation of a nucleic acid in the sample.In some embodiments, an in situ reverse transcription reaction is usedto synthesize cDNA from targeted mRNA in the tissue sample. In someembodiments, an in situ amplification reaction can be used to producemultiple genomic DNA amplicons from targeted genes in the tissue sample.In some embodiments, there is no in situ biochemistry step, andsynthesis of cDNA is performed after capture or extraction of the RNAfrom the tissue.

At a step 220, the target nucleic acid in the tissue section istransferred onto an array such that the position of a nucleic acid onthe array can be correlated with a position in the tissue section. Insome embodiments, the target nucleic acid comprises an mRNA. In someembodiments, the target nucleic acid comprises a cDNA synthesized insitu. In some embodiments, the nucleic acid comprises genomic DNAamplicons generated by in situ amplification. In some embodiments, thearray is an array of capture sites, such as capture sites 105 of capturearray 100 shown in FIG. 1A and FIG. 1B. In some embodiments, the arrayis an array of beads (e.g., universal capture beads 150 of FIG. 1C) thatinclude a plurality of capture probes. Other examples of an arrayinclude an array of wells or pores or projections or a sequencing flowcell that includes a plurality of capture probes. The nucleic acid canbe captured onto the array, for example, by hybridizing the nucleic acidto the capture probes on the array. In some embodiments, the nucleicacid can be captured onto the array by single-strand ligation of thenucleic acid onto universal adaptor oligonucleotides.

In this and other embodiments described herein, the probes may becontacted with target nucleic acid by placing the tissue directly on thesurface comprising the probes; placing the tissue on a substance such asa filter or a gel or a thin buffer layer separating the tissue from theprobes such that the target nucleic acid may diffuse from the tissue,through the substance to the probes; placing the tissue on a substance,such as a filter or a gel or a thin buffer layer separating the tissuefrom the probes such that the probes may diffuse from the surfacecomprising the probes, through the substance to the target nucleic acid;extracting the target nucleic acid from the tissue onto an intermediatesubstrate (e.g., a gel, filter, solid substrate, or combinations of theforegoing), which is then placed on the surface supporting the probes;and combinations of the foregoing. In each case, the technique isselected to substantially maintain information encoding the spatialorientation of the targets in the sample.

At a step 225, a sequencing library is prepared. In some embodiments,the sequencing library is prepared for sequencing-by-synthesis. Librarypreparation may be accomplished on the capture array substrate, or thenucleic acids may be cleaved from the substrate and pooled, so thatlibrary preparation can be accomplished separately, e.g., using aNeoPrep™ Library Prep System (Illumina, Inc., San Diego).

At a step 230, the library is sequenced. Sequencing may be accomplishedusing any sequencing technique. Examples of suitable sequencers includethose available from, or being developed by, Illumina, Inc., F.Hoffmann-La Roche AG, Life Technologies, Inc., Beckman Coulter, Inc.,Pacific Biosciences, Inc., Oxford Nanopore, Inc., and/or theiraffiliates.

At a step 235, the sequence data is analyzed (e.g., mutations and/orvariant calling) and the spatial information is decoded. The spatialinformation can be used to provide information as to the location of thenucleic acid in the tissue section.

(a) Hybridization-Based Capture of Nucleic Acid

In some embodiments, hybridization-based capture is used to capturetarget nucleic acids in a tissue sample onto capture probes on an array.The array may be, for example, an array of beads or wells or pores orprojections, a planar surface, or a sequencing flow cell that includes aplurality of capture probes. In one example, the tissue sample iscontacted with capture probes that are fixed on the surface of thearray. The tissue sample may be placed directly on the surfacecomprising the capture probes or the tissue sample may be placed on asubstance such as a filter or a gel or a thin buffer layer separatingthe tissue sample from the capture probes such that the target nucleicacid may diffuse from the tissue through the substance to the captureprobes.

In another example, the tissue sample is contacted with the array andthe capture probes on the array are released into the tissue sample forhybridization to the nucleic acids in the tissue sample. The tissuesample may be placed directly on the surface comprising the captureprobes or the tissue sample may be placed on a substance such as afilter or a gel or a thin buffer layer separating the tissue sample fromthe capture probes such that the released capture probes may diffusefrom the array through the substance to the nucleic acid in the tissuesample. The capture probes may be anchored on the array using areleasable group or a selectively cleavable portion or linker. Thecapture probes may be released from the array using, for example,chemical cleavage, enzymatic cleavage or photo-cleavage. In anotherexample, the capture probes may be printed onto the surface of the arrayand dried down. The capture probes may be released from the array byrehydration. In yet another example, the capture probes may be printedonto the array using a substance that dissolves in the presence of acertain treatment. The treatment to release the capture probes is thenapplied prior to the placement of the tissue sample onto the array.

In some embodiments, the nucleic acid is total mRNA. In someembodiments, the nucleic acid is gene-specific mRNA.

FIG. 3 illustrates a side view of another embodiment of a capture site105 of capture array 100 of FIG. 1A and shows an embodiment of a processof capturing total mRNA in a tissue sample onto the array. In thisembodiment, capture probe 115 comprises a poly-T capture region 310. Anucleic acid sample, such as a tissue sample or substrate comprisingnucleic acid targets derived from a tissue sample (not shown), thatincludes a plurality of mRNA molecules 315 is contacted with the capturesite 105. mRNA molecule 315 comprises a poly-A tail 320. A mutation 325can be present in mRNA molecule 315. Mutation 325 can, for example, be asingle nucleotide polymorphism (SNP). mRNA molecule 315 is captured oncapture probe 115 by hybridization of poly-A tail 320 to poly-T captureregion 310. Poly-T capture region 310 can also function as a reversetranscriptase primer for synthesis of first strand cDNA from capturedmRNA molecule 315 (indicated by dashed arrow).

In this example, the tissue sample is contacted with a capture probe 115that is fixed on the surface of capture site 105. mRNA molecule 315 iscaptured onto the array by hybridization to poly-T capture sequence 310.In another example (not shown), the tissue sample is contacted with thearray and capture probe 115 is released from capture site 105 into thetissue sample by cleavage of optional cleavable sequence 120.

In some embodiments (not shown), capture region 310 is a randomoligonucleotide (“randomer”) capture region that can be used to capturea random pool of RNA molecules. The random oligonucleotide captureregion can, for example, comprise a random sequence with reducedcomplexity such that capture of ribosomal RNA in the tissue sample issubstantially reduced.

FIG. 4 illustrates a side view of some embodiments of a capture site 105of capture array 100 of FIG. 1A and shows an embodiment of a process ofcapturing individually targeted (i.e., gene-specific) mRNA in a tissuesample onto the array. In this embodiment, two capture probes 115, i.e.,capture probe 115 a and 115 b, are shown. Capture probe 115 a comprisesa gene-specific capture region 410 a. Similarly, capture probe 115 bcomprises a different gene-specific capture region 410 b. A nucleic acidsample, such as a tissue sample or substrate comprising nucleic acidtargets derived from a tissue sample (not shown), that comprises aplurality of different mRNA molecules 415 is contacted with the capturesite 105. In this embodiment, a first mRNA molecule 415 a is thetranscript from a first gene and a second mRNA molecule 415 b is thetranscript from a second gene. mRNA molecule 415 a can include amutation 420 a. mRNA molecule 415 b can include a mutation 420 b.Mutations 420 can, for example, be SNPs. mRNA molecule 415 a is capturedon capture probe 115 a by hybridization of complementary mRNA sequencesto gene-specific capture region 410 a. Similarly, mRNA molecule 415 b iscaptured on capture probe 115 b by hybridization of a complementary mRNAmolecule to gene-specific capture region 410 b. Gene-specific captureregion 410 can also function as reverse transcriptase primers forsynthesis of first strand cDNA from captured mRNA molecule 415.

In this example, the tissue sample is contacted with capture probes 115that is fixed on the surface of capture site 105. mRNA molecules 415 arecaptured onto the array by hybridization to gene-specific capturesequences 410. In another example (not shown), the tissue sample iscontacted with the array and capture probes 115 are released fromcapture site 105 into the tissue sample by cleavage of optionalcleavable sequence 120.

In some embodiments, hybridization-based capture is used to capture cDNAgenerated by in situ reverse transcription of RNA in a tissue sampleonto an array.

FIG. 5 illustrates a flow diagram of an embodiment of a method 500 ofgenerating cDNA by in situ reverse transcription of RNA in a tissuesample for capture onto an array (e.g., capture array 100 of FIG. 1A).Method 500 comprises, but is not limited to, some or all of thefollowing steps.

At a step 510, gene-specific cDNA is synthesized from target mRNA in atissue sample by in situ reverse transcription. For example, anoligonucleotide sequence that comprises a first gene-specific primer anda universal capture sequence can be used to prime first strand cDNAsynthesis.

At an optional step 515, cDNA is amplified in the tissue sample by insitu isothermal amplification. For example, an oligonucleotide sequencethat comprises a second gene-specific primer and an SBS primer sequence,e.g., SBS12, can be used for isothermal amplification.

At a step 520, the amplified cDNA is captured onto an array. The cDNA iscaptured onto the array by hybridizing the cDNA to capture probes on thearray. In some embodiments the capture probes include a universalcapture sequence and can be used to capture cDNA synthesized using agene-specific primer that comprises a complementary capture sequence asdescribed in more detail with reference to FIG. 6. In some embodiments,the capture probes include gene-specific capture sequences and can beused to capture cDNA synthesized using a pool of gene-specific primersor random primers that include an SBS primer sequence as described inmore detail with reference to FIG. 7.

FIG. 6 illustrates an embodiment of the steps of a method 500 of FIG. 5.In this embodiment, an in situ isothermal amplification step (i.e.,optional step 515) is used prior to transfer of DNA to an array. Namely,a tissue section (not shown) comprises a target mRNA molecule 610.Target mRNA molecule 610 can include a mutation 615. At step 510, targetmRNA molecule 610 is reverse transcribed in situ using a reversetranscription (RT) primer 620. RT primer 620 comprises a firstgene-specific primer region 625 and a universal capture region 630. RTprimer 620 can also include a unique molecular identifier (UMI) region(not shown). A cDNA molecule 635 synthesized using RT primer 620comprises universal capture region 630. At step 515, cDNA molecule 635is amplified by in situ isothermal amplification using an amplificationprimer 640. Amplification primer 640 comprises a second gene-specificprimer region 645 and an SBS sequencing primer region 650 comprising,e.g., a SBS12 sequence. A DNA molecule 655 generated using amplificationprimer 640 comprises universal capture region 630 and SBS sequencingprimer 650. At step 520, DNA molecule 655 is captured onto the capturesite 105. For example, the tissue sample containing DNA molecule 655 ora substrate comprising DNA molecule 655 derived from the tissue sample(not shown) is contacted with the capture site 105. In some embodiments,capture probe 115 on the capture site 105 comprises a capture region 660that is complementary to universal capture region 630; SBS sequencingprimer 130 is SBS3. DNA molecule 655 is captured onto the capture site105 by hybridization of universal capture region 630 to capture region660.

In this example, both capture region 660 and universal capture region630 can also function as primers for an extension reaction. When captureregion 660 is used as a primer, mutation 615 in DNA molecule 655 iscopied. When universal capture region 630 is used as a primer, spatialaddress region 130 and SBS primer region 125 are copied. Both extensionproducts can be used for downstream library generation.

In this example, the tissue sample is contacted with capture probe 115that is fixed on the surface of capture site 105. DNA molecule 655 iscaptured onto the array by hybridization of universal capture region 630to capture region 660. In another example (not shown), the tissue sampleis contacted with the array and capture probes 115 are released fromcapture site 105 into the tissue sample by cleavage of optionalcleavable sequence 120.

FIG. 7 illustrates an embodiment of the steps of a method 500 of FIG. 5.In this embodiment, a gene-specific capture probe is used to capture aspecific cDNA and optional isothermal amplification step 515 of FIG. 5is omitted. Namely, a tissue section (not shown) comprises target mRNAmolecule 610. Target mRNA molecule 610 can include mutation 615. At step510, target mRNA molecule 610 is reverse transcribed in situ using an RTprimer 710. RT primer 710 comprises a gene-specific primer region 625and an SBS primer region 720, e.g., SBS3. RT primer 710 can also includea UMI sequence (not shown). A cDNA molecule 725 synthesized using RTprimer 710 comprises SBS primer region 720. At step 520, cDNA molecule725 is captured onto the capture site 105. For example, the tissuesample containing DNA molecule 725 or a substrate comprising DNAmolecule 725 derived from the tissue sample (not shown) is contactedwith the capture site 105. In this embodiment, capture probe 115 on thecapture site 105 comprises a gene-specific capture region 730 that iscomplementary to a sequences in the 3′ end of cDNA molecule 725; SBSprimer region 125 is SBS12. cDNA molecule 725 is captured on the capturesite 105 by hybridization of capture region 730 to complementarysequences in the 3′ end of cDNA molecule 725.

In some embodiments, hybridization-based capture can be used to transferamplicons generated by in situ amplification of genomic DNA in a tissuesample to an array. In some embodiments, genomic DNA amplicons aregenerated using a “TSCA-like” amplification approach. In someembodiments, genomic DNA amplicons are generated using a DNA-Padlockapproach.

FIG. 8 illustrates a flow diagram of an embodiment of a method 800 ofcapturing DNA amplicons onto an array (e.g., capture array 100 of FIG.1A), wherein the DNA amplicons are generated by in situ amplification oftarget nucleic acid. In this embodiment, the amplification reaction is aTSCA-like amplification (TruSeq Custom Amplicon assembly, Illumina).Method 800 comprises, but is not limited to, some or all of thefollowing steps.

At a step 810, a pair of gene-specific capture oligonucleotides thatflank a region of interest are hybridized in situ to genomic DNA. Forexample, a first capture oligonucleotide that hybridizes 5′ to a regionof interest can comprise a gene-specific sequence and a universalcapture sequence. A second capture oligonucleotide that hybridizes 3′ tothe region of interest can comprise a second gene-specific sequence andan SBS primer sequence (e.g., SBS12).

At a step 815, an in situ extension/ligation reaction is performedbetween the flanking capture oligonucleotides across the region ofinterest.

At a step 820, DNA flanked by capture oligonucleotides is amplified byin situ isothermal amplification to generate multiple copies of theregion of interest, i.e., multiple genomic amplicons. Isothermalamplification can be performed, for example, using primer sequences thatare complementary to the universal capture sequence and the SBS primersequence.

At a step 825, the genomic amplicons are transferred onto an array andcaptured by hybridization to universal capture regions on the array.

FIG. 9 illustrates an embodiment of the steps of a method 800 of FIG. 8.Namely, a tissue section (not shown) comprises a target genomic DNAmolecule 910. Target DNA molecule 910 can include a mutation 915. Atstep 810, a first gene-specific capture oligonucleotide 920 and a secondgene-specific capture oligonucleotide 925 that flank a region ofinterest are hybridized in situ to DNA molecule 910. Captureoligonucleotide 920 comprises a gene-specific region 930 and a universalcapture region 935. Capture oligonucleotide 920 can also comprise a UMIregion (not shown). Capture oligonucleotide 925 comprises a secondgene-specific region 940 and an SBS primer region 945 (e.g., SBS12). Atstep 815, an extension/ligation reaction is performed in situ betweenthe flanking capture oligonucleotides 920 and 925 across the region ofinterest. A DNA molecule 950 that is formed in the extension/ligationreaction comprises universal capture region 935 and SBS primer region945. At step 820, DNA molecule 950 is amplified by in situ isothermalamplification to generate multiple copies (multiple amplicons) of thetargeted region of interest. Isothermal amplification can be performed,for example, using a primer region 935 a that is complementary touniversal capture region 935 and a primer region 945 a that iscomplementary to SBS primer region 945. At step 825, genomic amplicons950 are captured onto the capture site 105. For example, the tissuesample containing genomic amplicons 950 or a substrate comprisinggenomic amplicons 950 derived from the tissue sample (not shown) iscontacted with the capture site 105. In this example, capture probe 115on the capture site 105 comprises a capture region 960 that iscomplementary to universal capture region 935; SBS primer region 130 isSBS3. DNA amplicons 950 are captured on the capture site 105 byhybridization of universal capture region 935 to capture region 960.

In this example, the tissue sample is contacted with capture probe 115that is fixed on the surface of capture site 105. Genomic amplicon 950is captured onto the array by hybridization of universal capturesequence 935 to capture sequence 960. In another example (not shown),the tissue sample is contacted with the array and capture probes 115 arereleased from capture site 105 into the tissue sample by cleavage ofoptional cleavable sequence 120.

FIG. 10 illustrates a flow diagram of embodiments of a method 1000 ofcapturing DNA amplicons onto an array (e.g., capture array 100 of FIG.1A), wherein the DNA amplicons are generated by in situ amplification oftarget nucleic acid. In some embodiment, the amplification reaction canbe a DNA padlock amplification. Method 1000 can comprise, but is notlimited to, some or all of the following steps.

At a step 1010, a padlock capture probe is hybridized in situ to genomicDNA. For example, the padlock capture probe can comprise a firstgene-specific sequence and a SBS primer sequence that are linked via alinker sequence to a universal capture sequence and a secondgene-specific sequence. The first gene-specific sequence and the secondgene-specific sequence flank a target region of interest in the genomicDNA.

At a step 1015, an in situ extension/ligation reaction is performedbetween the flanking gene-specific sequences in the padlock captureprobe across the targeted region to generate a circular molecule.

At a step 1020, DNA flanked by the padlock capture probe is amplified byin situ rolling circle amplification to generate a concatamer oftargeted amplicons. The rolling circle amplification can be performed,for example, using a primer sequence that is complementary to the SBSprimer sequence in the padlock capture probe.

At a step 1025, targeted amplicon concatamers are captured onto an arraythat comprises a universal capture sequence.

FIG. 11 illustrates the steps of a method 1000 of FIG. 10. Namely, atissue section (not shown) comprises a target genomic DNA molecule 1110.Target DNA molecule 1110 can include a mutation 1115. At step 1010, apadlock capture probe 1120 is hybridized in situ to DNA molecule 1110.Padlock capture probe 1120 can comprise a first gene-specific region1125 and an SBS primer region 1130 (e.g., SBS12) that can be linked viaa linker region 1135 to a universal capture region 1140 and a secondgene-specific region 1145. Padlock capture probe 1120 can also include aunique molecular identifier (UMI) region (not shown), to facilitatecorrection of sequencing errors. First gene-specific region 1125 andsecond gene-specific region 1145 flank a target region of interest inDNA molecule 1110. At a step 1015, an in situ extension/ligationreaction is performed between flanking first gene-specific region 1125and second gene-specific region 1145 across the targeted region ofinterest to form a circular molecule 1150. In some embodiments,CircLigase™ (Epicentre, Illumina) can be used to ligate circularmolecule 1150 prior to amplification. At a step 1020, circular molecule1150 is amplified by in situ rolling circle amplification to generate aconcatamer 1155 that comprises multiple copies of circular molecule1150. The rolling circle amplification is performed using a primersequence (not shown) that is complementary to SBS primer region 1130 oncircular molecule 1150. At step 1025, concatamer 1155 is captured ontothe capture site 105 that comprises cleavable region 120, SBS sequencingprimer region 125 (e.g., SBS3), spatial address region 130, and captureregion 135 (e.g., a universal capture sequence). For example, the tissuesample containing concatamer 1155 or a substrate comprising concatamer1155 derived from the tissue sample (not shown) is contacted with thecapture site 105. In some embodiments, capture probes 115 on capturesite 105 comprises a capture region 1160 that is complementary touniversal capture region 1140; SBS primer region comprises a SBS3sequence. Concatamer 1155 is captured on the capture site 105 byhybridization of universal capture regions 1140 to capture regions 1160.

In this example, the tissue sample is contacted with capture probe 115that is fixed on the surface of capture site 105. Concatamer 1155 iscaptured onto the array by hybridization of universal capture sequences1140 to capture sequence 1160. In another example (not shown), thetissue sample is contacted with the array and capture probes 115 arereleased from capture site 105 into the tissue sample by cleavage ofoptional cleavable sequence 120.

(b) Ligation-Based Capture of Nucleic Acid

In some embodiments, a ligation-based capture is used to capture targetnucleic acids in a tissue sample onto capture probes on an array. Thearray may be, for example, an array of beads or wells or pores orprojections, a planar surface, or a sequencing flow cell that includes aplurality of capture probes. In one example, the tissue sample iscontacted with capture probes that are fixed on the surface of thearray. The tissue sample may be placed directly on the surfacecomprising the capture probes or the tissue sample may be placed on asubstance such as a filter or a gel or a thin buffer layer separatingthe tissue sample from the capture probes such that the target nucleicacid may diffuse from the tissue through the substance to the captureprobes.

In another example, the tissue sample is contacted with the array andthe capture probes on the array are released into the tissue sample forhybridization to the nucleic acids in the tissue sample. The tissuesample may be placed directly on the surface comprising the captureprobes or the tissue sample may be placed on a substance such as afilter or a gel or a thin buffer layer separating the tissue sample fromthe capture probes such that the released capture probes may diffusefrom the array through the substance to the nucleic acid in the tissuesample. The capture probes may be anchored on the array using areleasable group or a selectively cleavable portion or linker. Thecapture probes may be released from the array using, for example,chemical cleavage, enzymatic cleavage or photo-cleavage. In anotherexample, the capture probes may be printed onto the surface of the arrayand dried down. The capture probes may be released from the array byrehydration. In yet another example, the capture probes may be printedonto the array using a substance that dissolves in the presence of acertain treatment. The treatment to release the capture probes is thenapplied prior to the placement of the tissue sample onto the array.

In one example, the nucleic acid is cDNA synthesized by in situ reversetranscription of RNA in a tissue sample. In another example, the nucleicacid is DNA amplicons generated by in situ amplification of genomic DNAin a tissue sample.

FIG. 12 illustrates a flow diagram of an embodiment of a method 1200 ofcapturing cDNA onto an array (e.g., capture array 100 of FIG. 1A) bysingle-strand ligation, wherein the cDNA is generated by in situ reversetranscription of target RNA molecules. Method 1200 comprises, but is notlimited to, some or all of the following steps.

At a step 1210, cDNA is synthesized in situ using gene-specific primers.For example, a gene-specific reverse transcription primer (RT) thatcomprises a first gene-specific primer region and a unique molecularidentifier (UMI) region can be used to prime first strand cDNAsynthesis. The cDNAcan be modified on the 5′ or 3′ end to preventself-ligation. In some embodiments, a modification to preventself-ligation of the cDNA can be pre-incorporated into the UMI sequenceprior to in situ cDNA synthesis. In some embodiments, a modificationsuch as the addition of a “tail” oligonucleotide region can be addedpost-cDNA synthesis to prevent self-ligation of the cDNA.

At a step 1215, the cDNA is transferred to a bead array and captured byligating the cDNA to universal adaptor oligonucleotides on the beadarray. The universal adaptor oligonucleotides can include a cleavableregion, a SBS primer sequence (e.g., SBS3), and a spatial address asdescribed with reference to FIG. 1A and FIG. 1C.

At a step 1220, the cDNA is cleaved from the bead array.

At a step 1225, second strand cDNA is synthesized using gene-specificprimers. The gene-specific primers can include, for example, agene-specific sequence and an SBS sequencing primer sequence (e.g.,SBS12).

At a step 1230, the cDNA is amplified using a pair of SBS primers togenerate a sequencing library. For example, a first SBS primer cancomprise SBS12 complementary sequences and P7 sequences. A second SBSprimer can comprise SBS3 complementary sequences and P5 sequences. Theresulting library amplicons can be flanked on the 5′ end by P7 sequenceand SBS primer sequences and by UMI, spatial address, SBS sequencingprimer, and P5 sequences on the 3′ end. P7 and P5 sequences can be usedto bind DNA amplicons to a flow cell surface for subsequent clusteramplification and sequencing.

At a step 1235, the library is sequenced.

FIGS. 13A and 13B illustrate the steps of a method 1200 of FIG. 12. Insome embodiments, cDNA synthesized in situ can be captured onto a beadarray that comprises universal capture bead 150 of FIG. 1C. Namely, atissue section (not shown) can comprise a target RNA molecule 1310.Target RNA molecule 1310 can include a mutation 1315. At step 1210, cDNAis synthesized in situ using a gene-specific RT primer 1320. RT primer1320 can comprise a gene-specific region 1325 and a UMI region 1330. AcDNA molecule 1335 synthesized using RT primer 1320 comprises UMI region1330. At step 1215, cDNA molecule 1335 is captured onto universalcapture bead 150. For example, the tissue sample containing cDNAmolecule 1335 or a substrate comprising cDNA molecule 1335 derived fromthe tissue sample (not shown) is contacted with universal capture bead150. cDNA molecule 1335 is captured on universal capture bead 150 byligation of UMI 1330 to spatial address region 130 in universal adaptoroligonucleotide 160. At step 1220, cDNA 1335 is cleaved from universalcapture bead 150. cDNA molecule 1335 now comprises SBS primer region 125(e.g., SBS3) and spatial address region 130. At step 1225, second strandcDNA is synthesized using a gene-specific primer 1340. Gene-specificprimer 1340 comprises a gene-specific region 1345 and a SBS primerregion 1350 (e.g., SBS12). A second strand cDNA molecule 1355synthesized using gene specific primer 1340 comprises SBS sequencingprimer region 1350, UMI region 1330, spatial address region 130, and SBSprimer region 125. At step 1230, cDNA molecule 1355 is amplified using afirst SBS primer 1360 and a second SBS primer 1365. SBS primer 1360comprises an SBS complementary region 1350 a that is complementary toSBS sequencing primer 1350 and a P7 primer region 1370. SBS primer 1365comprises an SBS complementary region 125 a that is complementary to SBSprimer region 125 and a P5 primer region 1375. A library amplicon 1380synthesized using SBS primers 1360 and 1365 is flanked on the 5′ end byP7 primer region 1370 and SBS primer region 1350 and on the 3′ end byUMI region 1330, spatial address region 130, SBS sequencing primerregion 125, and P5 primer region 1375. At step 1235 (not shown), thelibrary is sequenced.

In this example, the tissue sample is contacted with universal adaptoroligonucleotide 160 that is fixed on the surface of universal capturebead 150. cDNA molecule 1335 is captured onto the array by ligation ofUMI 1330 to spatial address 130 in universal adaptor oligonucleotide160. In another example (not shown), the tissue sample is contacted withthe array and universal adaptor oligonucleotides 160 are released fromthe array into the tissue sample by cleavage of optional cleavablesequence 120.

In some embodiments, an anchor PCR step (not shown) to enrich for targetcDNA sequences can be optionally performed prior to step 1225. Forexample, an anchor PCR amplification can be first performed usinggene-specific primers without SBS primer sequence 1350. Following theanchor PCR amplification step, a second amplification can be performedusing gene-specific primer 1340 that comprises gene-specific region 1345and SBS sequencing primer region 1350.

FIG. 14 illustrates a flow diagram of embodiments of a method 1400 ofcapturing DNA amplicons onto an array (e.g., capture array 100 of FIG.1A), wherein the DNA amplicons can be generated by in situ amplificationof target nucleic acid. Method 1400 can comprise, but is not limited to,some or all of the following steps.

At a step 1410, a pair of gene-specific capture probes that flank aregion(s) of interest are hybridized in situ to genomic DNA. Forexample, a first capture oligonucleotide that hybridizes 5′ to a regionof interest can comprise a first gene-specific sequence and a universalsequence. A second capture oligonucleotide that hybridizes 3′ to aregion of interest can comprise a second gene-specific sequence, a UMI,and a SBS primer sequence (e.g., SBS3).

At a step 1415, an in situ extension/ligation reaction is performedbetween the flanking capture probes across regions of interest.

At a step 1420, DNA flanked by capture probes is amplified by in situisothermal amplification to generate multiple copies of the regions ofinterest, i.e., multiple amplicons. Isothermal amplification can beperformed, for example, using primer sequences that are complementary tothe universal sequence and the SBS primer sequence.

At a step 1425, a 3′ tail oligonucleotide can be added onto the DNAamplicons to prevent self-ligation.

At a step 1430, DNA amplicons are transferred to a bead array (e.g., anarray of universal capture beads 150 shown in FIG. 1C) and captured byligation onto universal capture oligonucleotides on the bead array. Forexample, the DNA amplicons can be denatured, transferred to the beadarray and ligated onto universal capture oligonucleotides. The universalcapture oligonucleotides can include a cleavable sequence, an SBS primersequence (e.g., SBS12), and a spatial address sequence as describe withreference to FIG. 1C. Both strands (i.e., top strand and bottom strand)of the denatured amplicons can ligate to the capture oligonucleotides onthe bead array.

At a step 1435, DNA amplicons are cleaved from the bead array.

At a step 1440, DNA amplicons are amplified using a pair of SBS primersto generate a sequencing library. For example, a first SBS primer cancomprise SBS12 complementary sequences and P7 sequences. A second SBSprimer can comprise SBS3 complementary sequences and P5 sequences. Insome embodiments, only targeted sequences that are flanked by SBS12 andSBS3 sequences are amplified (i.e., the top strand of the DNA amplicon).The resulting library amplicons are flanked on the 5′ end by P7 andSBS12 primer sequences and by UMI, spatial address, SBS3 primer, and P5sequences on the 3′ end.

At a step 1445, the library is sequenced.

FIGS. 15A, 15B, and 15C illustrate the steps of a method 1400 of FIG.14. Namely, a tissue section (not shown) comprises a targeted DNA region1510. Target DNA molecule 1510 can include a mutation 1515. At step1410, a first gene-specific capture probe 1520 and a secondgene-specific capture probe 1525 that flank a region of interest arehybridized in situ to DNA molecule 1510. Gene-specific capture probe1520 comprises a gene-specific region 1530 and a universal region 1535.Gene-specific capture probe 1525 can comprise a second gene-specificregion 1540, a UMI region 1545, and an SBS primer region 1550 (e.g.,SBS3). At step 1415, an in situ extension/ligation reaction is performedbetween the flanking gene-specific capture probes 1520 and 1525 acrossthe region of interest to generate a DNA molecule 1555. DNA molecule1555 comprises universal region 1535, UMI region 1545, and SBS primerregion 1550. At step 1420, DNA 1555 is amplified (e.g., in situisothermal amplification) to generate multiple copies (i.e., multipleamplicons) of the targeted region of interest. Amplification, e.g.,isothermal amplification, is performed using a primer region 1535 a thatis complementary to universal region 1535 and a primer region 1550 athat is complementary to SBS primer region 1550. At step 1425, a 3′ tailoligonucleotide 1560 is added onto the DNA amplicons to preventself-ligation. At step 1430, DNA molecule 1555 is denatured andtransferred to universal capture bead 150. For example, the tissuesample containing DNA molecule 1555 or a substrate comprising DNAmolecule 1555 derived from the tissue sample (not shown) is contactedwith universal capture bead 150. Each strand (i.e., top strand “A” andbottom strand “B”) of DNA molecule 1555 is captured on universal capturebead 150 by ligation of 3′ tail oligonucleotide 1560 to spatial addressregion 130 in universal adaptor oligonucleotide 160. At Step 1435, DNAmolecules 1555 are cleaved from universal capture bead 150. Twodifferent configurations of DNA molecule 1555 are formed: A) a DNAmolecule 1565 that comprises (in the 3′ to 5′ direction) SBS primerregion 125 (e.g., SBS12), spatial address region 130, 3′ tailoligonucleotide 1560, universal region 1535, DNA molecule 1555, UMIregion 1545, and SBS primer region 1550 (e.g., SBS3) and B) a DNAmolecule 1570 that comprises (in the 3′ to 5′ direction) SBS primerregion 125 (e.g., SBS12), spatial address region 130, 3′ tailoligonucleotide 1560, SBS primer region 1550 (e.g., SBS3), UMI region1545, DNA molecule 1555, and universal region 1535. At step 1440, DNAmolecules 1565 (A) and 1570 (B) are amplified using a first SBS primer1575 and a second SBS primer 1580. First SBS primer 1575 comprises anSBS primer sequence 125 a that is complementary to SBS primer sequence125 and a P7 sequence 1585. Second SBS primer 1580 comprises a SBSprimer sequence 1550 a that is complementary to SBS primer sequence 1550and a P5 sequence. A library amplicon 1595 amplified from DNA molecule1565 using SBS primers 1575 and 1580 is flanked on the 3′ end by P7region 1585, SBS primer region 125 (e.g. SBS12), spatial address region130, 3′ tail oligonucleotide 1560, and universal region 1535 and on the5′ end by UMI region 1545, SBS primer region 1550 (e.g., SBS3), and P5primer region 1590. Because of the configuration of the SBS sequences inDNA molecule 1570, DNA molecule 1570 is not amplified.

In this example, the tissue sample is contacted with universal adaptoroligonucleotide 160 that is fixed on the surface of universal capturebead 150. DNA molecule 1555 is captured on universal capture bead 150 byligation of 3′ tail oligonucleotide 1560 to spatial address 130. Inanother example (not shown), the tissue sample is contacted with thearray and universal adaptor oligonucleotides 160 are released from thearray into the tissue sample by cleavage of optional cleavable sequence120.

4.3 Transfer of Nucleic Acids onto Capture Arrays

In some embodiments of the methods described herein nucleic acidmolecules can be transferred from a sample, such as a tissue section,onto a capture array by passive diffusion.

In some embodiments, the transfer of nucleic acid molecules from asample onto a capture array can be facilitated, e.g., throughelectrophoresis or centrifugation.

In some embodiments, nucleic acid molecules can be transferred directlyfrom a sample, such as a tissue section, onto a capture array. Forexample, a tissue section can be placed directly onto a capture array.

In some embodiments, the nucleic acid molecules can be transferredindirectly from a sample, such as a tissue section, onto a capturearray. For example, nucleic acids from a tissue section can betransferred first to one or more intermediate substrates, e.g., anysubstrate other than a capture array, such that the relative spatialorientation of nucleic acids on the intermediate substrate mirrors therelative spatial orientation in the tissue section. The nucleic acidscan then be transferred from the intermediate substrate to the capturearray, such that the relative spatial orientation of the nucleic acidson the capture array mirrors the relative spatial orientation of thenucleic acids in the tissue section. Indirect transfer can occur, e.g.,through passive diffusion or through facilitated transfer (e.g.,electrophoresis or centrifugation). The intermediate substrate can be,e.g., a membrane, such as a nylon membrane, or a gel, or a microwellplate. In some embodiments, the nucleic acids from a tissue sample canbe transferred first to a gel, then to one or more membranes, and thento the capture array. In some embodiments, the intermediate substratecan be configured such that it stabilizes the separation of nucleicacids in different spatial regions of the tissue section. For example,nucleic acids in different spatial regions in the tissue section can bepermanently separated from one another by placing different fragments ofthe tissue section (or of a gel or membrane comprising nucleic acidsfrom the tissue section) into different wells of the microwell platesuch that the relative spatial orientation of the tissue sectionfragments in the microwell plate can be correlated with the relativespatial orientation of the fragments in the tissue section. The nucleicacids in the tissue fragments in the microwell plate can subsequently betransferred from the microwell plate to the capture array.

In some embodiments, intermediate substrates can be used to produce twoor more copies of nucleic acids whose relative spatial orientation canbe correlated with their relative spatial orientation in a tissuesection. For example, nucleic acids can be transferred from a tissuesection onto several membranes, e.g., by placing the tissue section ontoa membrane that forms the first layer of several layered membranes.Transfer from the tissue section can onto the two or more layeredmembranes can occur, e.g., through passive diffusion or it can befacilitated. The spatial orientation of the nucleic acids on each of thetwo or more layered membranes corresponds to the relative spatialorientation of the nucleic acids in the tissue section. The nucleicacids on the two or more layered membranes can subsequently betransferred to two or more capture arrays.

In some embodiments, the transfer of nucleic acid molecules from asample onto a capture array can be facilitated using magneticallyresponsive nanoparticles

(a) Facilitated Transfer of Nucleic Acids onto Capture Arrays

In some embodiments, facilitated nucleic acid transfer can result ingreater yields of nucleic acid transfer from the sample onto the capturearray, compared to nucleic acid transfer by passive diffusion underotherwise comparable experimental condition (e.g., transfer temperature,transfer buffer, and the like). In some embodiments, facilitated nucleicacid transfer can result in at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, or at least 100-foldgreater yields compared to nucleic acid transfer by passive diffusion.Methods for analyzing the efficiency of nucleic acid transfer are wellknown in the art, for example using radioisotope-labeled orfluorescently-labeled nucleic acids, or comparing yields or efficienciesof next-generation sequencing reactions.

In some embodiments, facilitated nucleic acid transfer can allow for areduction of transfer times, compared to nucleic acid transfer bypassive diffusion under otherwise comparable experimental conditions(e.g., a reduction of transfer times from more than 12 h, more than 24h, more than 36 h, or more than 48 h to less than 6 h, less than 4 h,less than 2 h, or less than 1 h). In some embodiments, facilitatednucleic acid transfer can result in at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold reduction of transfertimes, compared to nucleic acid transfer by passive diffusion. Methodsfor analyzing or comparing transfer times are well known in the art. Forexample, the transfer time can represent the time required to transfer acertain amount of nucleic acid from the sample to the capture array, asdetermined, e.g., through the use of radioisotope-labeled orfluorescently-labeled nucleic acids, or by comparing yields orefficiencies of next-generation sequencing reactions.

In some embodiments, facilitated nucleic acid transfer can allow for thetransfer of nucleic acids from larger samples, e.g., thicker tissueslices, onto a capture array, compared to nucleic acid transfer bypassive diffusion under otherwise comparable experimental conditions. Insome embodiments, facilitated nucleic acid transfer can allow for thetransfer of nucleic acids from tissue slices having an at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, or at least 10-foldlarger thickness, compared to the thickness of samples typically appliedwhen transferring nucleic acids by passive diffusion (e.g., from about10 μm to about 100 μm in thickness). In some embodiments, the thicknessof a tissue slice can be less than about 5 μm.

In some embodiments, nucleic acid transfer from a tissue sample can befacilitated with respect to certain capture sites on a capture array,whereas the nucleic acid transfer from a tissue sample can occur throughpassive diffusion with respect to certain other capture sites on thecapture array. In some embodiments, nucleic acid transfer from a tissuesample onto a capture array can be facilitated with respect to aselected subset set capture sites, e.g., a subset of at least 1%, atleast 3%, at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at last 80%, atleast 90%, at least 95%, at least 98%, or at least 99% of capture sites.

(b) Electrophoretic System for Spatial Detection and Analysis of NucleicAcids in a Tissue Sample

In some embodiments, a capture array (i.e., an array of capture sites)can be integrated with an electrophoretic system to force nucleic acidmolecules to move directly from a tissue section onto capture probes. Insome embodiments, the nucleic acid is RNA. In some embodiments thenucleic acid is DNA (e.g., cDNA or DNA amplicons).

FIG. 16 illustrates a side view of a portion of an exemplaryelectrophoretic transfer system 1600 that is configured for spatialdetection and analysis of nucleic acid in a tissue sample.Electrophoretic transfer system 1600 comprises a capture array 1610.Capture array 1610 can be capture array 100 of FIG. 1A. Capture array1610 comprises a solid support 1615. In some embodiments, solid support1615 is a planar glass substrate. An arrangement (e.g., rows andcolumns) of capture sites 1620 are formed on solid support 1615. In someembodiments, a row of six capture sites 1620 are shown (i.e., capturesites 1620 a through 1620 f), but any number and configuration ofcapture sites 1620 can be used. A plurality of oligonucleotides (notshown) are immobilized at each of the capture sites 1620. Associatedwith each capture site 1620 is a bottom electrode 1625 (e.g., six bottomelectrodes 1625 a through 1625 f). In this example, one bottom electrode1625 for each capture site 1620 is shown, but any number of bottomelectrodes 1625 per capture site 1620 may be used. For example, capturearray 1610 may include 2 bottom electrodes 1625 per capture site 1620,or 10 bottom electrodes 1625 per capture site 1620, or 100 bottomelectrodes 1625 per capture site 1620, or any number of bottomelectrodes 1625 per capture site 1620. A sample substrate 1630 ispositioned atop capture array 1610. In one example, sample substrate1630 is a planar glass substrate. Sample substrate 1630 includes anarrangement of top electrodes 1635. In this example, the arrangement oftop electrodes 1635 corresponds to the arrangement of bottom electrodes1625 on capture array 1610, i.e., one top electrode 1635 (e.g., topelectrode s 1635 a through 1635 f) per bottom electrode 1625 (e.g.,bottom electrodes 1625 a through 1625 f). In another example (notshown), sample substrate 1630 includes a single top electrode 1635. Atissue sample 1640 is mounted on the surface of sample substrate 1630that is facing capture sites 1620 of capture array 1610.

Capture sites 1620, bottom electrodes 1625 and top electrodes 1635 areconfigured for electrophoretic transfer and capture of nucleic acidsfrom a tissue sample such that spatial orientation is maintained anddiffusion of nucleic acids from the tissue sample and loss of nucleicacids between capture sites 1620 is eliminated or substantially reduced.Each of the capture sites 1620 can be addressed (charged) individuallyor all or selected groups of the capture sites 1620 can be addressed incommon as a single unit. A voltage source 1645 is connected acrossbottom electrodes 1625 and top electrodes 1635. In the presence of anelectric field supplied by voltage source 1645, a plurality of nucleicacids 1650 are transferred from tissue sample 1640 to capture sites1620. Nucleic acids 1650 are captured at capture sites 1620 byhybridization to capture probes (not shown) that are immobilized atcapture sites 1620.

4.4 Spatial Detection and Analysis of Nucleic Acids in a Tissue SampleUsing Capture Probe Sets.

According to the methods described herein, spatial detection andanalysis of nucleic acids in a tissue sample can be performed using setsof two or more capture probes (e.g., 3 or more, 4 or more, 5 or more, 6or more, 7 or more, 8 or more, 9 or more, or 10 or more capture probes).Typically at least a first capture probe in a set of capture probes isimmobilized on a capture array. In some embodiments, a second captureprobe can be immobilized on the same capture array as the first captureprobe, e.g., in proximity to the first capture probe, e.g., in the samecapture site. In some embodiments, a second capture probe can beimmobilized on a particle, such as a magnetic particle or a magneticnanoparticle. See, e.g., Section 5.6. In some embodiments, a secondcapture probe can be in solution, e.g., to be used to perform in situreactions with a nucleic acid in a tissue sample.

The capture probes in the capture probe sets individually andindependently can have a variety of different regions, e.g., a captureregion (e.g., a universal or gene-specific capture region), a primerbinding region (e.g., a SBS primer region, such as a SBS3 or SBS12region, or another universal region, such as a P5 or P7 region), aspatial address region (e.g., a partial or combinatorial spatial addressregion), or a cleavable region.

In some embodiments, only one capture probe in a set of capture probescomprises a capture region. In some embodiments, two or more captureprobes in a set of capture probes comprise as capture region.

In some embodiments, only one probe in a set of capture probes comprisesa spatial address region, e.g., such as a complete spatial addressregion describing the position of a capture site on a capture array. Insome embodiments, two or more probes in a set of capture probes cancomprise a spatial address region, e.g., two or more probes can eachcomprise a partial spatial address region (i.e., combinatorial addressregion), wherein each partial address region describes the position of acapture site on a capture array, e.g., along the x-axis or the y-axis.

In some embodiments, a set of capture probes can comprise at least onecapture probe comprising a capture region and a spatial address region(e.g., a complete or a partial spatial address region). In someembodiments, no capture probe in a set of capture probes comprises botha capture region and a spatial address region.

In another aspect, provided herein is a capture array for spatialdetection and analysis of nucleic acids in a tissue sample, comprising acapture site comprising a set of capture probes. In some embodiments,the set of capture probes comprises at least two capture probes (i.e.,at least a pair of capture probes). In some embodiments, the set ofcapture probes comprises three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, or ten or morecapture probes. In some embodiments, the capture array can be integratedin an electrophoretic transfer system described herein. See, e.g.,Section 5.3.

FIG. 17 illustrates a side view of one capture site 1700 on a capturearray (e.g., capture array 100 of FIG. 1A), wherein the one capture site1700 comprises two separate sets of immobilized capture probes. In someembodiments, a set of immobilized capture probes comprises two captureprobes (i.e., a pair of capture probes). In some embodiments, a set ofimmobilized capture probes comprises three or more capture probes. Byway of example, FIG. 17 shows capture site 1700 a of a capture array. Afirst set of capture probes 1710 comprises an SBS primer region 1715(e.g., SBS3) and a spatial address region 1720. Each capture probe 1710immobilized at capture site 1700 a comprises the same unique spatialaddress region 1720. Capture probes 1710 immobilized at other capturesites 1700 (not shown), e.g., capture sites 1700 b through 1700 f, eachinclude their own unique spatial address region 1720 (e.g., spatialaddress region 1720 b through 1720 f), i.e., each capture site 1700 hasa unique spatial address region. A second set of capture probes 1725comprises a second SBS primer region 1730 (e.g., SBS12) and agene-specific capture region 1735, e.g., gene-specific capture region1735 a and gene-specific capture region 1735 b. In some embodiments, thesecond set of capture probes does not comprise spatial address sequence.In this example, two spatially addressed capture probes 1710 and captureprobes 1725 are shown, but any number of spatially addressed captureprobes 1710 and capture probes 1725 can be immobilized at capture site1700 a. In some embodiments, the capture array illustrated in FIG. 17can be integrated in an electrophoretic transfer system 1600 of FIG. 16.

Because at least two different sets of capture probes are used, e.g., inthe embodiment illustrated in FIG. 17, the number of oligonucleotidesrequired to achieve gene-specific capture is substantially reduced. Forexample, in a conventional approach, to RNA from 100 different genes at20,000 capture sites, 2 million different spatially addressed captureoligonucleotides would be required. However, according to the methodsdescribed herein, to capture RNA from 100 different genes at 20,000capture sites, only 20,000 spatially addressed oligonucleotides and 100capture oligonucleotides are required.

In some embodiments, a capture array comprises a capture site (e.g.,1700 a) comprising a pair of capture probes immobilized on a surface(e.g., 1710 and 1725 a), wherein a first capture probe (e.g., 1710) ofthe pair of capture probes comprises a first primer binding region(e.g., SBS primer binding region 1715; e.g., SBS3) and a spatial addressregion (e.g., spatial address region 1720), and wherein a second captureprobe (e.g., 1725 a) of the pair of capture probes comprises a secondprimer binding region (e.g., SBS primer binding region 1730; e.g.,SBS12) and a capture region (e.g., 1735 a).

In some embodiments, the first capture probe does not comprise agene-specific region.

In some embodiments, the second capture probe does not comprise aspatial address region.

In some embodiments, the capture site is a plurality of capture sites.In some embodiments, the plurality of capture sites is 2 or more, 10 ormore, 30 or more, 100 or more, 300 or more, 1,000 or more, 3,000 ormore, 10,000 or more, 30,000 or more, 100,000 or more, 300,000 or more,1,000,000 or more 3,000,000 or more, or 10,000,000 or 1,000,000,000 ormore capture sites.

In some embodiments, the capture array comprises a capture site densityof 1 or more, 2 or more, 10 or more, 30 or more, 100 or more, 300 ormore, 1,000 or more, 3,000 or more, 10,000 or more, 100,000 or more,1,000,000 or more, capture sites per square centimeter (cm²).

In some embodiments, the pair of capture probes in a capture site is aplurality of pairs of capture probes. In some embodiments, the pluralityof capture probes is 2 or more, 10 or more, 30 or more, 100 or more, 300or more, 1,000 or more, 3,000 or more, 10,000 or more, 30,000 or more,100,000 or more, 300,000 or more, 1,000,000 or more 3,000,000 or more,or 10,000,000 or more, 100,000,000 or more, or 1,000,000,000 or morecapture probes.

In some embodiments, the pair of capture probes in a capture site of acapture array is a plurality of pairs of capture probes. In someembodiments, each first capture probe in the plurality of pairs ofcapture probes within the same capture site comprises the same spatialaddress sequence. In some embodiments, each first capture probe in theplurality of pairs of capture probes in different capture sitescomprises a different spatial address sequence.

In some embodiments, one or more capture sites of capture array have thesame number of first capture probes and of second capture probes. Insome embodiments, one or more capture sites have more first captureprobes than second capture probes. For example, in some embodiments, oneor more capture sites have at least 2-fold, at least 3-fold, at least10-fold, at least 30-fold, at least 100-fold, at least 300-fold, atleast 1,000-fold, at least 3,000-fold, or at least 10,000-fold morefirst capture probes than second capture probes. In some embodiments,one or more capture sites have more second capture probes than firstcapture probes. For example, in some embodiments, one or more capturesites have at least 2-fold, at least 3-fold, at least 10-fold, at least30-fold, at least 100-fold, at least 300-fold, at least 1,000-fold, atleast 3,000-fold, or at least 10,000-fold more second capture probesthan first capture probes.

In some embodiments, the capture array is integrated into anelectrophoresis system. In some embodiments, the electrophoresis systemis an electrophoresis system as described in Section 5.3 (see, e.g.,FIG. 16). In some embodiments, each capture site is independentlyelectrically addressable in the electrophoresis system. In someembodiments, a capture site in the electrophoresis system is configuredfor transfer and capture of nucleic acids from a tissue sample such thatdiffusion of nucleic acids from the tissue sample and loss of nucleicacids between capture sites are substantially reduced relative to apassive nucleic acid transfer occurring under otherwise identicalconditions in the absence of the electrophoresis system, e.g., bydiffusion.

In some embodiments, the surface of the capture array is a planarsurface, e.g., a glass surface. See, e.g., FIG. 1B. In some embodiments,the surface of the capture array comprises one or more wells. In someembodiments, the one or more wells correspond to one or more capturesites. In some embodiments, the surface of the capture array is a beadsurface, e.g., as illustrated in FIG. 1C.

In some embodiments, the capture region in the second capture probe is agene-specific capture region. In some embodiments, the gene-specificcapture region in the second capture probe comprises the sequence of aTruSeq™ Custom Amplicon (TSCA) oligonucleotide probe (Illumina, Inc.).For example, the gene-specific capture regions in a plurality of secondcapture probes in a capture site can comprise a plurality of sequencesof TSCA oligonucleotide probes.

In some embodiments, the capture region in the second capture probe is auniversal capture region. In some embodiments, the universal captureregion in the second capture probe comprises a random primer sequence.For example, the capture regions in a plurality of second capture probesin a capture site can comprise randomized sequences. In someembodiments, the universal capture region in a second capture probecomprises a poly-T capture sequence. For example, some or all of theuniversal capture sequences in a plurality of second capture probes in acapture site can comprise a poly-T capture sequence.

In some embodiments, the capture regions in one or more second captureprobes of a capture site can be essentially the same capture regions intwo or more capture sites of the capture array. In some embodiments, atleast 1%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 99% ofsecond capture probes have the same capture regions in two or morecapture sites of the capture array (e.g., in at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% of capture sites on a capturearray).

In some embodiments, the capture regions in the one or more secondcapture probes of a capture array can be essentially the same capturesequences in at least 1%, at least 5%, at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99% of capture sites on a capture array. In some embodiments, thecapture regions in the one or more second capture probes of a capturearray can be essentially the same capture sequences in essentially allcapture sites on a capture array.

In some embodiments, the spatial address region comprises two or morepartial spatial address regions (e.g., a first, a second, and,optionally, a third partial address region) that can be combined in acombinatorial manner (e.g., X*Y*Z). In some embodiments, the spatialaddress region comprises a first and a second partial address region toidentify the position of a capture site on the capture array in thefirst (X) and second (Y) dimension. In some embodiments, the spatialaddress region further comprises a third partial address region toidentify the location of a tissue slice (and of a nucleic acidtransferred from the tissue slice) in the tissue sample in the third (Z)dimension.

In some embodiments, the first or second capture probe on the capturearray further comprises a temporal address region (T) to identify therelative sequence of timepoints at which a sample was obtained in thecourse of a time-course experiment (e.g., a time-course experiment todetermine changes of gene-expressions in a tissue over time in responseto a chemical, biological, or physical stimulus).

In some embodiments, two or more address regions (e.g., spatial ortemporal address regions) in a capture probe are consecutive. In someembodiments, two or more address regions are separated by one or morenucleic acids (e.g., by 2 or more, 3 or more, 10 or more, 30 or more,100 or more, 300 or more, or 1,000 or more nucleic acids).

FIG. 18 illustrates a flow diagram of an embodiment of a method 1800 oftransferring nucleic acids from a tissue sample to a capture array forgeneration of a spatially addressed sequencing library, wherein thecapture array comprises capture sites that include separate pairs ofimmobilized capture probes, e.g., as shown in FIG. 17. In thisembodiment, the nucleic acid is RNA. In some embodiments of the methodillustrated in FIG. 18, the capture array is integrated into anelectrophoretic transfer system, such as the electrophoretic transfersystem 1600 of FIG. 16. Method 1800 comprises, but is not limited to,some or all of the following steps.

Optionally, at a step 1810, a nucleic acid from a tissue sample can beelectrophoretically transferred to a capture array. For example and nowreferring to FIG. 16 and FIG. 17, sample substrate 1630 with tissuesample 1640 thereon can be placed atop capture array 1610. In thisexample, capture sites 1620 of capture array 1610 include separate pairsof immobilized capture probes, e.g., as shown in FIG. 17. An electricfield is applied to capture sites 1620. As capture sites 1620 areactivated, nucleic acids 1650 from tissue sample 1640 are transferred tocapture array 1610 and hybridize to capture probes 1725 that areimmobilized at capture sites 1620. In this embodiment, capture probes1725 are gene-specific capture probes designed to capture specificmRNAs.

In some embodiments, at step 1810, the transfer of a nucleic acid from asample, such as the tissue sample 1640, can occur by passive diffusion.In some embodiments, at step 1810, the transfer of a nucleic acid from asample, such as the tissue sample 1640, is facilitated by a method otherthan electrophoresis.

At a step 1815, first strand cDNA is synthesized. For example,gene-specific capture regions 1735 can function as reverse transcriptaseprimers for synthesis of first strand cDNA from captured mRNA molecules.

At a step 1820, first strand cDNA is covalently linked to the secondcapture probe 1710 by single-strand ligation of cDNA to spatial addressregion 1720.

At a step 1825, second strand cDNA is synthesized using a primer that iscomplementary to SBS primer region 1715.

At a step 1830, second strand cDNA molecules are released from capturesite 1700 by denaturation.

At a step 1835, the cDNA is amplified to generate a sequencing library.FIGS. 19A, 19B, 19C, and 19D show pictorially the steps of method 1800of FIG. 18. Namely, at step 1810, a plurality of mRNA molecules 1910 aretransferred (e.g., electrophoretically) from a tissue section (notshown) onto capture site 1700 and hybridize to capture probes 1725. Insome embodiments, a first mRNA molecule 1910 a is a transcript from afirst gene and a second mRNA molecule 1910 b is a transcript from asecond gene. mRNA molecule 1910 a can include a mutation 1915 a. mRNAmolecule 1910 b can include a mutation 1915 b. mRNA molecule 1910 a iscaptured on capture probe 1725 a by hybridization of complementary mRNAsequences to gene-specific capture region 1735 a. Similarly, mRNAmolecule 1910 b is captured on capture probe 1725 b by hybridization ofcomplementary mRNA sequences to gene-specific capture region 1735 b.

At step 1815, first strand cDNA is synthesized using gene-specificcapture region 1735 as a primer. A cDNA molecule 1920 (i.e., cDNAmolecules 1920 a and 1920 b) include SBS primer region 1730.

At step 1820, cDNA molecule 1920 is covalently linked to spatiallyaddressed oligonucleotides 1710 by single-strand ligation of cDNAmolecule 1920 to spatial address region 1720.

At step 1825, second strand cDNA is synthesized using a primer region1715 a that is complementary to SBS primer region 1715.

At step 1830, second strand cDNA molecules 1920 are released fromcapture site 1700 by denaturation. cDNA molecules 1920 now include SBSprimer region 1715, spatial address region 1720, and SBS primer region1730.

At step 1835, cDNA molecules 1920 are amplified to generate a sequencinglibrary. In a first amplification reaction, cDNA molecules are amplifiedusing a first SBS primer 1925. SBS primer 1925 comprises an SBScomplementary region 1730 a that is complementary to SBS primer region1730 and a P7 region 1930. Amplicons 1935 (i.e., amplicons 1935 a and1935 b) are flanked on the 5′ end by P7 region 1930 and SBS primerregion 1730 and on the 3′ end by spatial address region 1720 and SBSprimer region 1715. Amplicons 1935 are amplified using a second SBSprimer 1940. SBS primer 1940 comprises an SBS complementary region 1715a that is complementary to SBS primer region 1715 and a P5 region 1945.Amplicons 1935 (i.e., amplicons 1935 a and 1935 b) are now flanked by P7region 1930 and SBS primer region 1730 and by spatial address region1720, SBS primer region 1715, and P5 region 1945.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing acapture array described herein. In some embodiments, the capture arraycomprises a capture site comprising a set of capture probes. In someembodiments, the set of capture probes comprises two capture probes(i.e., a pair of capture probes). In some embodiments, the set ofcapture probes comprises three or more, four or more, five or more, sixor more, seven or more, eight or more, nine or more, or ten or morecapture probes. In some embodiments, the capture array can be integratedin an electrophoretic transfer system described herein. See, e.g.,Section 5.3.

In some embodiments, the method comprises (a) providing a capture array,comprising a capture site comprising a pair of capture probes (e.g.,1710 and 1725 a) immobilized on a surface, wherein a first capture probeof the pair of capture probes comprises a first primer binding region(e.g., 1715) and a spatial address region (e.g., 1720), and wherein asecond capture probe of the pair of capture probes comprises a secondprimer binding region (e.g., 1730) and a capture region (e.g., 1735 a).

In some embodiments, the first capture probe does not comprise a captureregion.

In some embodiments, the second capture probe does not comprise aspatial address region.

In some embodiments, the method further comprises any one or more of thefollowing steps: (b) contacting the capture array with a tissue samplesuch that the position of a capture site on the array can be correlatedwith a position in the tissue sample; (c) allowing nucleic acids of thetissue sample to hybridize to the capture region of the second captureprobe; (d) extending the capture region of the second capture probe toform an immobilized first complementary strand of the nucleic acidhybridized to the gene-specific sequence; (e) ligating the immobilizedfirst complementary strand to the spatial address sequence of a firstcapture probe to immobilize the first complementary strand at both ends;(f) synthesizing a second complementary strand using a primercomplementary to the first primer binding sequence of the first captureprobe; (f) releasing the second complementary strand from the surface ofthe capture array; (g) analyzing the sequence of the released secondcomplementary strand, and (h) correlating the sequence of the releasedsecond complementary stand to the position of the nucleic acid in thetissue sample.

In some embodiments, allowing nucleic acids of the tissue sample tohybridize to the capture region of the second capture probe comprises anelectrophoretic transfer of the nucleic acids from the tissue sampleonto the capture array.

In some embodiments, allowing the nucleic acids of the tissue sample tohybridize to the capture region of the second capture probe comprisespassive diffusion of the nucleic acids from the tissue sample onto thecapture array.

In some embodiments, analyzing the sequence of the releasedcomplementary strand comprises next-generation sequencing, e.g., bysequencing-by-synthesis.

In some embodiments, the nucleic acids of the tissue sample comprise amessenger ribonucleic acid (mRNA).

In some embodiments, the first or second primer binding region comprisesa SBS primer sequence. In some embodiments, the SBS primer sequence is aSBS3 or SBS12 sequence.

In some embodiments, the capture region of the second capture probecomprises a single nucleotide variation (SNV). In some embodiments, themethod has a sensitivity of SNV detection of at least 0.00025% SNV(1/400,000 cells). The sensitivity of spatial NGS for detection ofsingle nucleotide variations is described in more detail with referenceto Table 3 hereinbelow.

In some embodiments, the capture region in the second capture probe is auniversal capture region. In some embodiments, the universal captureregion in the second capture probe comprises a random primer sequence.In some embodiments, the capture regions in the plurality of secondcapture probes in a capture site comprise 10 or more, 100 or more, 1,000or more, 10,000 or more, 100,000 or more, or 1,000,000 or morerandomized capture sequences. In some embodiments, the universal captureregion in the second capture probe comprises a poly-T capture sequence.

In some embodiments, the capture region in the second capture probe is agene-specific capture region. In some embodiments, the gene-specificcapture region in the second capture probe comprises the sequence of aTSCA oligonucleotide probe. In some embodiments, the capture regions inthe plurality of second capture probes in a capture site comprise 10 ormore, 100 or more, 1,000 or more, 10,000 or more, 100,000 or more, or1,000,000 or more TSCA capture sequences.

In some embodiments, at least one capture probe in a set of captureprobes is in solution, e.g., to hybridize with a nucleic acid in thetissue sample. FIG. 20 shows an exemplary embodiment of a process 2000of capturing a nucleic acid in a tissue sample for subsequent anchoringonto an array. In this embodiment, a capture probe 2010 comprises anucleic acid capture region 2015 and a universal array capture region2020. In one example, nucleic acid capture region 2015 is a randomprimer sequence. In another example, nucleic acid capture region 2015 isa gene-specific primer sequence. Universal array capture region 2020 isa universal sequence that is used to anchor a nucleic acid molecule ontoa capture array. Capture probe 2010 can be used in a solution-basedhybridization reaction to capture a nucleic acid molecule 2025 in atissue section. In one example, nucleic acid molecule 2025 is a genomicDNA molecule. Capture probe 2010 hybridizes to nucleic acid molecule2025 from a tissue sample and is extended to form a nucleic acidcomplementary to nucleic acid molecule 2025 (indicated by the arrow) andincorporate universal array capture region 2020 into the complementarystrand. Universal array capture region 2020 is then used to anchor thecopied nucleic acid molecule onto a capture array (not shown). Inanother example, nucleic acid molecule 2025 is an RNA molecule. Captureprobe 2010 hybridizes to nucleic acid molecule 2025 and is used as aprimer in a reverse transcription reaction to synthesize first strandcDNA (indicated by the arrow) and incorporate universal array captureregion 2020 into cDNA molecule. Universal array capture region 2020 isthen used to anchor the cDNA molecule onto a capture array (not shown).

In some embodiments, the method comprises (a) providing a capture array,comprising a capture site comprising a first capture probe immobilizedon a surface, wherein the capture probe comprises a cleavable region, afirst primer binding region and a spatial address region.

In some embodiments, the first capture probe does not comprise a captureregion.

In some embodiments, the method further comprises one or more of thefollowing steps: (b) contacting a tissue sample with a second captureprobe, wherein the second capture probe comprises a second primerbinding region and a capture region (e.g., a gene specific or auniversal capture region); (c) allowing nucleic acids of the tissuesample to hybridize to the capture region of the second capture probe;(d) extending the capture region of the second capture probe to form afirst complementary strand of the nucleic acid hybridized to the nucleicacid.

In some embodiments, the second capture probe does not comprise aspatial address region.

In some embodiments, the method further comprises one or more of thefollowing steps: (e) optionally, hybridizing a complementaryoligonucleotide to the spatial address region of the capture probe toform a double-stranded spatial address region; (f) contacting thecapture array with the tissue sample comprising the first complementarystrand of the nucleic acid such that the position of a capture site onthe array can be correlated with a position in the tissue sample; (g)allowing the first complementary strand of the nucleic acid to transferfrom the tissue sample onto the capture array; (h) ligating the firstcomplementary strand of the nucleic acid (that is optionally hybridizedto the nucleic acid) to the first capture probe (e.g., by blunt endligation, such as double stranded blunt end ligation) to form aspatially tagged double stranded nucleic acid comprising first andsecond primer binding sites and a cleavable domain; (g) releasing thedouble stranded nucleic acid from the surface of the capture array; (h)analyzing the sequence of the released double stranded nucleic acid, and(i) correlating the sequence of the released nucleic acid to theposition of the nucleic acid in the tissue sample.

In some embodiments, allowing the first complementary strand of thenucleic acid to transfer from the tissue sample onto the capture arraycomprises an electrophoretic transfer of the nucleic acids from thetissue sample onto the capture array.

In some embodiments, allowing the first complementary strand of thenucleic acid to transfer from the tissue sample onto the capture arraycomprises passive diffusion of the nucleic acids from the tissue sampleonto the capture array.

In some embodiments, of the released double stranded nucleic acidcomprises next-generation sequencing, e.g., by sequencing-by-synthesis.

In some embodiments, the nucleic acids of the tissue sample comprise amessenger ribonucleic acid (mRNA).

In some embodiments, the first or second primer binding region comprisesa SBS primer sequence. In some embodiments, the SBS primer sequence is aSBS3 or SBS12 sequence.

4.5 Combinatorial Indexing System

In another embodiment, a combinatorial indexing (addressing) system isused to provide spatial information for analysis of nucleic acids in atissue sample. In this approach, two or more spatial address sequencesare incorporated into a nucleic acid during preparation of a sequencinglibrary. A first spatial address is used to define a certain position(i.e., capture site) in the X dimension on a capture array and a secondspatial address sequence is used define a position (i.e., a capturesite) in the Y dimension on the capture array. During librarysequencing, both X and Y spatial address sequences are determined andthe sequence information is analyzed to define the specific position onthe capture array.

In one example, the tissue sample is contacted with capture probes thatare fixed on the surface of the array. The tissue sample may be placeddirectly on the surface comprising the capture probes or the tissuesample may be placed on a substance such as a filter or a gel or a thinbuffer layer separating the tissue sample from the capture probes suchthat the target nucleic acid may diffuse from the tissue through thesubstance to the capture probes.

FIGS. 21A and 21B show a grid array 2100 of a one-dimensional indexingscheme and a grid array 2105 of a two-dimensional indexing scheme,respectively, for spatial detection and analysis of nucleic acids in atissue sample. Referring to FIG. 21A, to spatially address 100 positionsin a one-dimensional indexing scheme, 100 unique spatial addresses arerequired. Referring now to FIG. 21B, to spatially address 100 positionsin a two-dimensional indexing scheme, 10 unique spatial addresses arerequired for the X dimension and 10 unique spatial addresses arerequired for the Y dimension, i.e., the total unique spatial addresssequences required is 20. Combinatorial indexing substantially reducesthe number of spatial addresses that are needed for spatial detectionand analysis of nucleic acids in a tissue sample.

In some embodiments, a capture site on an array comprises a firstcapture probe with a first spatial address sequence for the X dimensionand a second capture probe with a second spatial address sequence forthe Y dimension. In some embodiments, the first and second captureprobes are oriented in opposite directions on the capture site such thatboth the 5′ and 3′ ends of a RNA molecule are captured. Duringsubsequent library preparation steps, the first and second spatialaddress sequences are incorporated into library amplicons as describedin more detail with reference to FIG. 22 and FIGS. 23A and 23B.

In some embodiments, the first and second capture probes are oriented inthe same direction on the capture site such that only the 3′ end of amRNA molecule is captured. During subsequent library preparation steps,the first and second spatial address sequences are incorporated intolibrary amplicons as described in more detail with reference to FIG. 24and FIGS. 25A and 25B.

In some embodiments, two or more partial address region (e.g., a firstand a second partial address region) can be incorporated into a singlecapture probe. The two or more partial address regions can form aconsecutive region or be separated by one or more nucleic acids. Forexample, two or more partial address regions can be incorporated into asingle first capture probe of a capture probe pair, according to thecapture arrays and methods illustrated, e.g., in FIGS. 17-19.

FIG. 22 illustrates a flow diagram of an example of a method 2200 ofusing a combinatorial indexing system for generation of a spatiallyaddressed cDNA sequencing library. Method 2200 comprises, but is notlimited to, some or all of the following steps.

At a step 2210, a tissue sample that comprises a plurality of mRNAmolecules is contacted with a capture array. The capture array can be,for example, capture array 100 of FIG. 1A. For each gene-specific (i.e.,specifically targeted) mRNA molecule two capture probes are used, i.e.,a first capture probe that comprises sequences specific for the 3′ endof the mRNA molecule and a second capture probe that comprises sequencesspecific for the 5′ end of the mRNA molecule. mRNA molecules arecaptured onto the array by hybridization of mRNA to the gene-specificcapture regions on the capture probes. An example of the capture probeson capture array 100 are described in more detail with reference to FIG.23A.

At a step 2215, first strand cDNA is synthesized. For example,gene-specific capture regions on the first capture probe are used asreverse transcriptase primers for synthesis of first strand cDNA fromcaptured mRNA molecules. The cDNA molecule is then ligated to the 5′ endof the second capture probe.

At a step 2220, the cDNA molecule is released from the capture array.For example, the cDNA molecule is released from the capture array usinga cleavage reaction.

At a step 2225, the cDNA is amplified to generate a sequencing library.

FIGS. 23A and 23B illustrate the steps of method 2200 of FIG. 22. Inthis example, a single capture site 105 of capture array 100 of FIG. 1Ais shown. Capture site 105 comprises a first capture probe 2310 a and asecond capture probe 2310 b. Capture probes 2310 include a cleavabledomain 2315, an SBS primer region 2320, a spatial address region 2325, aunique molecular identifier (UMI) 2330, and a capture region 2335. Forexample, capture probe 2310 a comprises cleavable region 2315; SBSprimer region 2320 a, which comprises, e.g., a SBS3 sequence; spatialaddress region 2325 a, which comprises a unique spatial address sequencefor the X dimension; UMI region 2330 a, which comprises a uniquesequence for capture probe 2310 a; and a gene-specific capture region2335 a, which is specific for the 3′ end of a mRNA molecule. Captureprobe 2320 a is immobilized at capture site 105 in the 5′ to 3′orientation (i.e., the 5′ end of capture probe 2320 a is attached to thesurface of capture site 105). Similarly, capture probe 2310 b comprisescleavable sequence 2315; SBS primer region 2320 b, which comprises aSBS12 sequence; spatial address region 2325 b, which comprises a uniquespatial address region for the Y dimension; UMI region 2330 b, whichcomprises a unique sequence for capture probe 2310 b; and agene-specific capture region 2335 b, which is specific for the 5′ end ofthe mRNA molecule. Capture probe 2320 b is immobilized at capture site105 in the 3′ to 5′ orientation (i.e., the 3′ end of capture probe 2320a is attached to the surface of capture site 105).

At step 2210, a mRNA molecule 2340 in a tissue sample is captured oncapture probes 2310. For example, the tissue sample containing mRNAmolecule 2340 or a substrate comprising mRNA molecule 2340 derived fromthe tissue sample (not shown) is contacted with capture probes 2310.mRNA molecule 2340 can include a mutation 2345. mRNA molecule 2340 iscaptured at capture site 105 by hybridization of the 3′ end of mRNAmolecule 2340 to capture region 2335 a and hybridization of the 5′ endof mRNA molecule 2340 to capture region 2335 b.

At step 2215, a cDNA molecule 2350 is synthesized using capture region2335 a as a primer in a reverse transcription reaction. The 3′ end ofcDNA molecule 2350 is then ligated to the 5′ end of capture region 2335b.

At step 2220, cDNA molecule 2350 is released from capture site 105 bycleavage of cleavable region 2315. cDNA molecule 2350 comprises SBSprimer region 2320 a (i.e., SBS3), spatial address region 2325 a (i.e.,unique spatial address for the X dimension), UMI region 2330 a, UMIregion 2330 b, spatial address region 2325 b (i.e., unique spatialaddress for the Y dimension), and SBS primer region 2320 b (i.e.,SBS12).

At step 2225, cDNA molecule 2350 is amplified using an SBS primer 2355and a second SBS primer 2360. SBS primer 2355 comprises an SBS sequence2320 a′ that is complementary to SBS primer region 2320 a and a P5region 2365. SBS primer 2360 comprises an SBS region 2320 b′ that iscomplementary to SBS primer region 2320 b and a P7 region 2370. Alibrary amplicon 2375 synthesized using SBS primers 2355 and 2360 isflanked on the 5′ end by P5 region 2365, SBS primer region 2320 a (i.e.,SBS3), spatial address region 2325 a (i.e., unique spatial address forthe X dimension), UMI region 2330 a and on the 3′ end by UMI region 2330b, spatial address region 2325 b (i.e., unique spatial address for the Ydimension), SBS primer region 2320 b (i.e., SBS12), and P7 region 2370.

FIG. 24 illustrates a flow diagram of an example of an alternativemethod 2400 of using a combinatorial indexing system for generation of aspatially addressed cDNA sequencing library. Method 2400 comprises, butis not limited to, some or all of the following steps.

At a step 2410, a tissue sample that comprises a plurality of mRNAmolecules is contacted with a capture array. The capture array can be,for example, capture array 100 of FIG. 1A. For each gene-specific mRNAmolecule two capture probes are used, i.e., a first capture probe thatcomprises sequences specific for the 3′ end of the mRNA molecule and asecond capture probe that comprises sequences specific for the 3′ end ofa corresponding first strand cDNA molecule. mRNA molecules are capturedonto the array by hybridization of the 3′ end of the mRNA togene-specific regions on the first capture probe. An example of thecapture probes on capture array 100 are described in more detail withreference to FIG. 25A.

At a step 2415, first strand cDNA is synthesized. For example,gene-specific capture regions on the first capture probe are used asreverse transcriptase primers for synthesis of first strand cDNA fromcaptured mRNA molecules.

At a step 2420, first strand cDNA is captured on the second captureprobe. First strand cDNA is captured on the second capture probe byhybridization of the 3′ end of the cDNA to a gene-specific region on thesecond capture probe.

At a step 2425, second strand cDNA is synthesized. For example,gene-specific regions on the second capture probes are used as primersfor synthesis of second strand DNA molecules.

At a step 2430, second strand cDNA molecules are released from thecapture array. For example, cDNA molecules are released from the capturearray using a cleavage reaction.

At a step 2435, cDNA molecules are amplified to generate a sequencinglibrary.

FIGS. 25A, 25B, and 25C illustrate the steps of method 2400 of FIG. 24.In this example, a single capture site 105 of capture array 100 of FIG.1A is shown. Capture site 105 is essentially the same as described abovewith reference to FIG. 23A except that capture probes 2310 are orientedin the same direction at capture site 105, i.e., both capture probe 2310a and capture probe 2310 b are immobilized at capture site 105 in the 5′to 3′ direction (i.e., the 5′ end of capture probes 2310 a and 2310 bare attached to the surface of capture site 105). Capture region 2335 ais specific for the 3′ end of a mRNA molecule and capture region 2335 bis specific for the 3′ end of the corresponding first strand cDNAmolecule.

At step 2410, a mRNA molecule 2510 in a tissue sample is captured oncapture probe 2310 a. For example, the tissue sample containing mRNAmolecule 2510 or a substrate comprising mRNA molecule 2510 derived fromthe tissue sample (not shown) is contacted with capture probes 2310.mRNA molecule 2510 can include a mutation 2515. mRNA molecule 2510 iscaptured at capture site 105 by hybridization of the 3′ end of mRNAmolecule 2510 to capture region 2335 a.

At step 2415, first strand cDNA is synthesized using capture region 2335a as a primer in a reverse transcription reaction.

At step 2420, a first strand cDNA molecule 2520 is captured on captureprobe 2310 b by hybridization of the 3′ end of cDNA molecule 2520 tocapture region 2335 b.

At step 2425, second strand cDNA is synthesized in an extension reactionusing capture region 2335 b as a primer.

At step 2430, a cDNA molecule 2525 is released from capture site 105 bycleavage of cleavable region 2315. cDNA molecule 2525 comprises SBSprimer region 2320 a (i.e., SBS3), spatial address region 2325 a (i.e.,unique spatial address for the X dimension), UMI region 2330 a, UMIregion 2330 b, spatial address region 2325 b (i.e., unique spatialaddress for the Y dimension), and SBS primer region 2320 b (i.e.,SBS12).

At step 2435, cDNA molecule 2525 is amplified using an SBS primer 2530and a second SBS primer 2535. SBS primer 2530 comprises an SBS region2320 a′ that is complementary to SBS primer region 2320 a and a P5region 2540. SBS primer 2535 comprises an SBS region 2320 b′ that iscomplementary to SBS primer region 2320 b and a P7 region 2545. Alibrary amplicon 2550 synthesized using SBS primers 2530 and 2535 isflanked on the 5′ end by P5 region 2540, SBS primer region 2320 a (i.e.,SBS3), spatial address region 2325 a (i.e., unique spatial address forthe X dimension), UMI region 2330 a and on the 3′ end by UMI region 2330b, spatial address region 2325 b (i.e., unique spatial address for the Ydimension), SBS primer region 2320 b (i.e., SBS12), and P7 region 2545.

In some embodiments, a combinatorial indexing system can involve use oftwo different arrays, i.e., a first array that comprises spatial addresssequences for the X dimension and a second array that comprises spatialaddress sequences for the Y dimension. In one example, a first array canbe used to deliver reverse transcription (RT) primers to a tissue samplefor in situ synthesis of cDNA and a second array is used to capture thecDNA for generation of a spatially addressed sequencing library.

FIG. 26A illustrates a plan view of an example of an array 2600 fordelivery of RT primers to a tissue sample for in situ synthesis of cDNA.Array 2600 comprises an arrangement, e.g., rows, of delivery sites 2605on a solid support 2610. In this example, 10 delivery sites 2605 (e.g.,delivery sites 2605 a through 2605 j) are arranged on solid support2610. In one example, solid support 2610 is a glass coverslip. Aplurality of RT primers 2615 is deposited (e.g., printed) in a stripealong the length of each delivery site 2605. RT primers 2615 aredeposited at each delivery site 2605 such that they can be readilyreleased from array 2600 onto a tissue sample.

FIG. 26B illustrates a side view of a portion of one delivery site 2605of array 2600, wherein the portion of delivery site 2605 comprises atleast one RT primer 2615 for synthesis of cDNA from mRNA in a tissuesample. In this example, a single RT primer 2615 is shown, but anynumber of RT primers 2615 can be deposited on solid support 2610 at eachdelivery site 2605. RT primer 2615 comprises an SBS primer region 2620(e.g., SBS3), a spatial address region 2625 (i.e., unique spatialaddress for the X dimension), and a gene-specific primer region 2630. RTprimer 2615 has a unique spatial address region 2625 for each deliverysite 2605, i.e., delivery site 2605 a has a unique spatial addressregion 2625 a, delivery site 2605 b has a unique spatial address region2625 b, etc. Gene-specific primer region 2630 can be the samegene-specific region or they can be different gene-specific sequences.

FIG. 27A illustrates a plan view of an example of a capture array 2700for the capture of cDNA synthesized in situ using RT primers 2615 ofFIG. 26B. Capture array 2700 comprises an arrangement, e.g., columns, ofcapture sites 2705 on a solid support 2710. In this example, 10 capturesites 2705 (e.g., capture sites 2705 a through 2705 j) are arranged onsolid support 2710. In one example, solid support 2710 is a planar glasssurface. A plurality of capture probes 2715 are deposited in a stripealong the length of each capture site 2705.

FIG. 27B illustrates a side view of a portion of one capture site 2705of capture array 2700, wherein the portion of capture site 2705comprises at least one capture probe 2715 for capture of cDNAsynthesized in situ using RT primers 2615 of FIG. 26B. In this example,a single capture probe 2715 is shown, but any number of capture probes2715 can be immobilized on solid support 2710 at each capture site 2705.Capture probe 2715 comprises a cleavable region 2720, an SBS primerregion 2725 (e.g., SBS12), a spatial address region 2730, a uniquemolecular identifier (UMI) region 2735, and a gene-specific captureregion 2740. Capture probe 2715 has a unique spatial address region 2730for each capture site 2705, i.e., capture site 2705 a has a uniquespatial address region 2730 a, capture site 2705 b has a unique spatialaddress region 2730 b, etc. Capture region 2740 is complementary tocapture region 2630 of capture probe 2615 of FIG. 26B.

FIG. 28 illustrates a flow diagram of an example of a method 2800 ofgenerating a spatially addressed sequencing library, wherein a firstarray is used for in situ synthesis of first strand cDNA and a secondarray is used to capture the cDNA for subsequent library generation.Method 2800 comprises, but is not limited to, the following steps.

At a step 2810, a tissue sample that comprises a plurality of mRNAmolecules is contacted with a first array. The array is, for example,array 2600 of FIG. 26A that comprises a plurality of RT primers 2615. RTprimers 2615 are released from array 2600 onto the tissue sample.

At a step 2815, first strand cDNA is synthesized in situ. For example,gene-specific primer sequences on the RT primers are used to prime firststrand cDNA from targeted mRNA molecules in the tissue sample. Afterfirst strand cDNA synthesis, array 2600 is removed from the surface ofthe tissue sample.

At a step 2820, first strand cDNA is captured onto a second array andsecond strand cDNA is synthesized. For example, capture array 2700 ofFIG. 27A that comprises a plurality of capture probes 2715 is contactedwith the tissue sample. First strand cDNA is captured onto capture array2700 by hybridization of the 3′ end of the cDNA to gene-specific regionson capture probes 2715. Second strand cDNA is synthesized usinggene-specific regions on capture probe 2715 as primers in an extensionreaction.

At a step 2825, second strand cDNA molecules are released from thecapture array. For example, cDNA molecules are released from the capturearray using a cleavage reaction.

At a step 2830, cDNA molecules are amplified to generate a sequencinglibrary.

FIGS. 29A and 29B illustrate the steps of method 2800 of FIG. 28. Atstep 2810, a tissue sample (not shown) that comprises a mRNA molecule2910 is contacted with array 2600 (not shown). For example, the tissuesample containing mRNA molecule 2910 or a substrate comprising mRNAmolecule 2910 derived from the tissue sample (not shown) is contactedwith array 2600. mRNA molecule 2910 can include a mutation 2915. RTprimers 2615 are released from array 2600 onto the tissue sample andhybridize to mRNA molecule 2910 via gene-specific region 2630.

At step 2815, a first strand cDNA molecule 2920 is synthesized in situusing gene-specific region 2630 as a primer in a reverse transcriptionreaction.

At step 2820, capture array 2700 of FIG. 27A that comprises a pluralityof capture probes 2715 is contacted with the tissue sample. First strandcDNA 2920 is captured at capture site 2705 by hybridization of the 3′end of first strand cDNA 2920 to gene-specific capture region 2740.Second strand cDNA is synthesized in an extension reaction usinggene-specific capture region 2740 as a primer.

At step 2825, a second strand cDNA 2925 is released from capture site2705 by cleavage of cleavable region 2720. cDNA molecule 2925 comprisesSBS primer region 2725 (SBS12), spatial address region 2730 (i.e.,unique spatial address for the Y dimension), UMI region 2735, spatialaddress region 2625 (i.e., unique spatial address for the X dimension),and SBS primer region 2620 (SBS3).

At step 2830, cDNA molecule 2925 is amplified using an SBS primer 2930and an SBS primer 2935. SBS primer 2930 comprises an SBS region 2725 athat is complementary to SBS primer region 2725 and a P5 region 2940.SBS primer 2935 comprises an SBS region 2620 a that is complementary toSBS primer region 2620 and a P7 region 2945. A library amplicon 2950synthesized using SBS primers 2930 and 2935 is flanked on the 5′ end byP5 region 2940, SBS primer region 2725 (i.e., SBS3), spatial addressregion 2730 (i.e., unique spatial address for the Y dimension), UMIregion 2735 and on the 3′ end by spatial address region 2625 (i.e.,unique spatial address for the X dimension), SBS primer region 2620(i.e., SBS3), and P7 region 2945.

In this example, the tissue sample is contacted with capture probes 2715that are fixed on the surface of capture sites 2705. First strand cDNA2920 is captured at capture site 2705 by hybridization of the 3′ end offirst strand cDNA 2920 to gene-specific capture sequence 2740. Inanother example (not shown), the tissue sample is contacted with capturearray 2700 and capture probes 2715 are released from capture sites 2705into the tissue sample by cleavage of cleavable sequence 2720. Secondstrand cDNA is synthesized in an extension reaction using gene-specificcapture sequence 2740 as a primer.

4.5 Spatial Detection and Analysis of Nucleic Acid in a Tissue SampleUsing Releasable Capture Probes

In other embodiments, a spatially addressed array is used to releasecapture probes into a tissue section for generation of a spatiallyaddressed sequencing library. In this approach, spatially addressedcapture probes are deposited on the surface of a substrate (e.g., aglass coverslip) at distinct capture sites or “features.” In oneexample, the spatially addressed capture probes are anchored onto thesurface of the substrate by the formation of a cleavable bond. Thespatially addressed capture probes are released into a tissue section bycleavage of the reversible bond and incorporated into the nucleic acidin subsequent biochemical processing steps. In some embodiments, thespatially addressed capture probes are deposited on the substratesuspended in a matrix such as a BioGel matrix. The spatially addressedcapture probes suspended in the BioGel are released into a tissuesection, for example, by application of a heat treatment or a chemicaltreatment. Immobilizing spatially addressed capture probes on asubstrate surface using a cleavable bond or BioGel suspension obviatesthe need to capture nucleic acid (i.e., RNA, cDNA or genomic DNA) from atissue section onto a substrate surface for generation of a spatiallyaddressed library.

In one example, a spatially addressed capture probe comprises a randomprimer sequence that is used for in situ synthesis of cDNA from totalRNA in a tissue sample.

FIG. 30 illustrates a flow diagram of an example of a method 3000 ofgenerating a spatially addressed cDNA library using releasable captureprobes. Method 3000 comprises, but is not limited to, the followingsteps.

At a step 3010, a coverslip is printed with spatially addressed captureprobes to form an array of spatial features. In one example, thespatially addressed capture probes are printed on a 2 cm×2 cm coverslipto form an array of spatial features that are 100 μm in diameter with apitch of 35 μm. The spatially addressed capture probes include a randomprimer sequence for synthesis of cDNA in a reverse transcriptionreaction, a spatial address sequence, and a biotinylated SBS primersequence as described in more detail with reference to FIGS. 31A and31B. The spatially addressed capture probes also include a modificationat the 5′ end of the molecule for reversible attachment to thecoverslip. In one example, the spatially addressed capture probesinclude a 5′ disulfide modification as described in more detail withreference to FIGS. 32A, 32B, and 32C. In some embodiments, the spatiallyaddressed capture probes include a 5′ photocleavable linker as describedin more detail with reference to FIGS. 33A, 33B, and 33C.

At a step 3015, the coverslip is placed atop a semi-permeabilized FFPEtissue section mounted on a glass slide, such that the surface of thecoverslip with the spatially addressed capture probes thereon is incontact with the tissue section.

At a step 3020, the spatially addressed capture probes immobilized onthe coverslip are released from the surface of the coverslip into thecellular space of the tissue section. In one example, spatiallyaddressed capture probes that include a 5′ disulfide modification arereleased by flowing a solution of dithiothreitol (DTT) through thesemi-permeabilized tissue section. In some embodiments, spatiallyaddressed capture probes that include a 5′ photocleavable linker arereleased using ultraviolet light irradiation.

At a step 3025, first strand cDNA is synthesized in situ using a reversetranscription reaction. For example, a reverse-transcription master mixsolution is flowed between the coverslip and glass slide into thesemi-permeabilized tissue section. The coverslip acts as a barrier toprevent evaporation during the reaction. Because of the internal biotinlabel in the spatially addressed capture probes used in the reversetranscription reaction, first strand cDNA is biotinylated.

At a step 3030, RNA:cDNA hybrids are dissociated and the cellular matrixdisrupted. In one example, the RNA:cDNA duplexes are dissociated and thecellular matrix disrupted using a NaOH solution. In some embodiments,RNA:cDNA duplexes are dissociated and the cellular matrix disruptedusing a heat treatment protocol.

At a step 3035, the semi-permeabilized tissue sample with RNA and cDNAtherein is removed from the surface of glass slide and collected in acollection tube. In one example, the semi-permeabilized tissue samplewith RNA and first strand cDNA therein is removed from the glass slideby scraping into an Eppendorf tube. In some embodiments, thesemi-permeabilized tissue sample with RNA and first strand cDNA thereinis removed from the glass slide by placing the slide into a 50 mLcentrifuge tube and centrifuging to collect the material in a receptacleat the bottom of the tube.

At a step 3040, the biotinylated first strand cDNA is purified using tworounds of a streptavidin bead-based purification protocol. The purifiedfirst strand cDNA is collected in a PCR tube for subsequent processingsteps. The cDNA molecule comprises an SBS primer sequence (e.g., SBS12),a spatial address sequence, and the random primer sequence.

At a step 3045, first strand cDNA is amplified in a multiplex reactionusing a mix of forward and reverse primer pairs that flank, for example,one or more targeted SNVs. For example, a forward primer comprises agene-specific sequence that targets a SNV of interest, an SBS primersequence (e.g., SBS3), and P5 sequences. The gene-specific sequence isdesigned to be about 50 bp upstream of a targeted SNV. A reverse primercomprises SBS12 complementary sequences and P7 sequences.

At a step 3050, library amplicons are sequenced. For example, read 1(e.g., from about 50 bp to about 75 bp) of an SBS reaction providessequence information for the targeted SNV and read 2 (about 25 bp)provides sequence information for the spatial address.

FIGS. 31A and 31B illustrate schematic diagrams of a spatially addressedcapture probe 3100 that comprises a 5′ disulfide modification and aspatially addressed capture probe 3150 that comprises a 5′photocleavable linker, respectively. Referring to FIG. 31A, spatiallyaddressed capture probe 3100 comprises a random primer region 3110, aspatial address region 3115, and an SBS primer region 3120 (e.g.,SBS12). Random primer region 3110 can comprise, e.g., a 6 bp sequencethat can be used to prime cDNA synthesis from the entire transcriptomeof a cell in a reverse transcription reaction. Spatial address region3115 comprises a unique sequence for each spatial feature on an array.SBS primer region 3120 comprises an internal biotin label 3125. Internalbiotin label 3125 is used to purify the cDNA in subsequent processingsteps. Spatially addressed capture probe 3100 also comprises a 5′disulfide modification 3130. Disulfide modification 3130 is used toreversibly anchor spatially addressed capture probe 3100 onto thesurface of a glass substrate (e.g., glass coverslip) as described inmore detail with reference to FIGS. 32A, 32B, and 32C.

Referring to FIG. 31B, spatially addressed capture probe 3150 issubstantially the same as spatially addressed capture probe 3100 exceptthat the 5′ end of spatially addressed capture probe 3150 is modifiedwith a photocleavable amino linker 3155. Photocleavable amino linker3155 is used to reversibly anchor spatially addressed capture probe 3150onto the surface of a glass substrate (e.g., glass coverslip) asdescribed in more detail with reference to FIGS. 33A, 33B, and 33C.

FIGS. 32A, 32B, and 32C illustrate an example of a process of reversiblyanchoring spatially addressed capture probe 3100 of FIG. 31A onto thesurface of a glass coverslip.

In a first step and referring now to FIG. 32A, a glass coverslip 3210functionalized with a plurality of thiol (SH) groups 3215 is provided.Thiol functionalized coverslips are commercially available.

In a next step and referring now to FIG. 32B, spatially addressedcapture probes 3100 are deposited (e.g., printed) onto the surface ofglass coverslip 3210.

In a next step and referring now to FIG. 32C, spatially addressedcapture probes 3100 are anchored onto the surface of glass coverslip3210 by formation of a disulfide bond in a thiol-disulfide exchangereaction between thiol groups 3215 and disulfide modifications 3130 onspatially addressed capture probes 3100. Spatially addressed captureprobes 3100 can be subsequently released from glass coverslip 3210 bycleavage of the disulfide bond using a reducing agent. In one example,dithiothreital (DTT) is used to cleave the disulfide bond and releasespatially addressed capture probes 3100.

FIGS. 33A, 33B, and 33C illustrate an example of a process of reversiblyanchoring spatially addressed capture probes 3150 of FIG. 31B onto thesurface of a glass coverslip.

In a first step and referring now to FIG. 33A, a glass coverslip 3310functionalized with a plurality of aldehyde groups 3315 is provided.Aldehyde functionalized glass substrates are commercially available.

In a next step and referring now to FIG. 33B, spatially addressedcapture probes 3150 are deposited onto the surface of glass coverslip3310.

In a next step and referring now to FIG. 33C, spatially addressedcapture probes 3150 are anchored onto the surface of glass coverslip3310 by formation of a covalent bond between aldehyde groups 3315 andphotocleavable amino linkers 3155 on spatially addressed capture probes3150. Spatially addressed capture probes 3150 can be subsequentlyreleased from glass coverslip 3310 by cleavage of the covalent bondusing ultraviolet light irradiation (e.g., 350 nm for about 5 minutes).

FIGS. 34A and 34B show pictorially the steps of method 3000 of FIG. 30.At step 3010, a coverslip 3410 is printed with spatially addressedcapture probes (not shown) to form an array of spatial features 3415. Inone example, the spatially addressed capture probes are printed on a 2cm×2 cm coverslip to form an array of spatial features 3415 that are 100μm in diameter with a pitch of 35 μm. The spatially addressed captureprobes are, for example, spatially addressed capture probe 3100 of FIG.31 that are used to capture the entire transcriptome of a cell.

At step 3015, coverslip 3410 is placed atop a semi-permeabilized tissuesection 3420 that is mounted on a glass slide 3425. Tissue section 3420comprises a plurality of cells 3430. Each cell contains one or more RNAmolecules 3435. One or more RNA molecules 3435 can include a singlenucleotide variation (SNV) 3440.

At step 3020, the spatially addressed capture probes (indicated byarrows) immobilized on coverslip 3410 are released from the surface ofthe coverslip into the cellular space.

At step 3025, first strand cDNA is synthesized in situ in a reversetranscription reaction using random primer sequences 3110 on spatiallyaddressed capture probe 3100. For example, a reverse-transcriptionmaster mix solution (not shown) is flowed between coverslip 3410 andglass slide 3425 into semi-permeabilized tissue section 3420.

At step 3030, RNA:cDNA hybrids are dissociated and the cellular matrixin tissue sample 3420 is disrupted to release a cDNA molecule 3445. Inone example, the RNA:cDNA duplexes are dissociated and the cellularmatrix disrupted using a NaOH solution. In some embodiments, RNA:cDNAduplexes are dissociated and the cellular matrix disrupted using a heattreatment protocol.

At step 3035, semi-permeabilized tissue sample 3420 with cells 3430, RNAmolecules 3435, and cDNA molecules 3445 therein are removed from thesurface of glass slide 3425 and collected in a collection tube 3450. Inone example, semi-permeabilized tissue sample 3420 with cells 3430, RNAmolecules 3435, and cDNA molecules 3445 therein are removed from glassslide 3425 by scraping into an Eppendorf tube. In some embodiments,semi-permeabilized tissue sample 3420 with cells 3430, RNA molecules3435, and cDNA molecules 3445 therein are removed from glass slide 3425by placing glass slide 3425 into a 50 mL centrifuge tube andcentrifuging to collect the material in a receptacle at the bottom ofthe 50 mL tube.

At step 3040, biotinylated cDNA molecules 3445 are purified using tworounds of a streptavidin bead-based purification protocol.

At step 3045, cDNA molecules 3445 are amplified in a multiplex reactionusing a mix of forward and reverse primer pairs that flank targetedSNVs. For example, a forward primer 3455 comprises a gene-specificregion 3460 that targets a SNV (e.g., SNV 3440) of interest, an SBSprimer region 3465 (e.g., SBS3), and a P5 region 3470. Gene-specificregion 3460 is designed to be about 50 bp upstream of a targeted SNV. Areverse primer 3475 comprises an SBS12 complementary region 3120 a and aP7 region 3480. A library amplicon 3485 synthesized using forward primer3455 and reverse primer 3475 comprises P5 region 3470, SBS primer region3465, SNV 3440, random primer region 3110, spatial address region 3115,SBS primer region 3120, and P7 region 3480.

At step 3050, library amplicons are sequenced. For example, read 1(e.g., from about 50 bp to about 75 bp) of an SBS reaction providessequence information for the targeted SNV and read 2 (about 25 bp)provides sequence information for the spatial address.

In some embodiments, a spatially addressed capture probe comprisessequences for in situ targeted capture and amplification of genomic DNAin a tissue sample. In one example, the capture and amplification oftargeted genomic DNA regions is performed using a TSCA-like approach(TruSeq Custom Amplicon assembly, Illumina). In the TSCA-like approach,a pair of capture probes that flank a targeted region of interest (e.g.,an SNV) is used to capture genomic DNA. FIG. 35 illustrates a schematicdiagram of an example of a capture probe pair 3500 for capturing agenomic DNA region of interest. Capture probe pair 3500 comprises afirst capture probe 3510 that hybridizes 5′ to a region of interest anda second capture probe 3515 that hybridizes 3′ to the region ofinterest. First capture probe 3510 comprises an SBS primer region 3520 a(e.g., SBS12), a spatial address region 3525, a gene-specific region3530 a. Second capture probe 3515 comprises an SBS primer region 3520 b(e.g., SBS3) and a gene-specific region 3530 b.

FIG. 36 illustrates a flow diagram of an example of a method 3600 ofgenerating a spatially addressed genomic amplicon library usingreleasable capture probes. In this example, the capture andamplification of targeted genomic DNA regions is performed using aTSCA-like approach (TruSeq Custom Amplicon assembly, Illumina). Method3600 comprises, but is not limited to, the following steps.

At a step 3610, a coverslip with spatially addressed capture probesthereon is placed atop a semi-permeabilized FFPE tissue section mountedon a glass slide. In one example, the spatially addressed capture probesare suspended in a BioGel matrix that is deposited onto the surface ofthe coverslip. The spatially addressed capture probes are a pair ofprobes that include DNA sequences that flank a region of interest (e.g.,a SNV) in the genomic DNA. The capture probes also include sequences forsubsequent PCR amplification (e.g., SBS3 and SBS12 sequences) asdescribed above with reference to FIG. 35. One (or both) of the captureprobes can also include an internal biotin label for subsequentpurification of the targeted DNA region. The capture probes are releasedfrom the BioGel matrix onto the tissue section using, for example, aheat treatment protocol.

At a step 3615, the capture probes are hybridized to genomic DNA and anin situ extension/ligation reaction is performed between the flankingcapture probes across the targeted region of interest.

At a step 3620, the extension/ligation products are purified. Forexample, the tissue sample with extension/ligation products therein isremoved from the surface of the glass slide and collected in acollection tube. The extension/ligation products are then purified usingone or more rounds of a purification protocol, such as a streptavidinbead-based purification protocol.

At a step 3625, the extension/ligation products are PCR amplified to addindices and sequencing primers.

At a step 3630, library amplicons are sequenced.

FIG. 37 illustrates the steps of method 3600 of FIG. 36. At step 3610, acoverslip 3710 with spatially addressed capture probes in a BioGelmatrix thereon is placed atop a semi-permeabilized FFPE tissue section3715 mounted on a glass slide 3720. Tissue section 3715 comprises a cell3725. Cell 3725 comprises a region of targeted genomic DNA 3730. GenomicDNA 3730 can include a SNV 3735. First capture probe 3510 and secondcapture probe 3515 are released into tissue section 3715 using a heattreatment protocol.

At step 3615, first capture probe 3510 and second capture probe 3515 arehybridized to genomic DNA and an in situ extension/ligation reaction isperformed between the flanking capture probes across the targeted regionof interest to generate an extension/ligation product 3740.

At step 3620, the extension/ligation product 3740 is purified. Forexample, the tissue sample with extension/ligation product 3740 thereinis removed from the surface of the glass slide and collected in acollection tube 3745. Extension/ligation product 3740 is then purifiedusing one or more rounds of a purification protocol, such as astreptavidin bead-based purification protocol.

At step 3625, extension/ligation product 3740 is PCR amplified using aforward primer 3750 and a reverse primer 3755 to add sequencingadapters. Forward primer 3750 comprises an SBS region 3520 b′ that iscomplementary to SBS primer region 3520 b and a P5 region 3760. Reverseprimer 3755 comprises an SBS region 3520 a′ that is complementary to3520 a and a P7 region 3765.

4.6 Particle-Based Capture of Nucleic Acids

In some embodiments, nucleic acids in tissue samples can be firstcaptured by probes immobilized on particles, such as nanoparticles, andthen transferred to a capture array described herein. The particle basedtransfer of nucleic acids can increase the efficiency, e.g., the yieldor the kinetics, of the nucleic acid transfer from the tissue sample tothe capture array.

Nucleic acids can be transferred from a tissue sample to a capture arrayby transferring particles comprising the nucleic acids from the tissuesample to the capture array. Particle transfer can be facilitated, e.g.,by using magnetically responsive particles, such as magneticallyresponsive nanoparticles, and by applying a magnetic field to the tissuesample and the capture array to facilitate the transfer of themagnetically responsive nanoparticles from the tissue sample to thecapture array. In some embodiments, particle transfer from the tissuesample to the capture array can be facilitated, e.g., by using amolecular interaction, such as a ligand-binding interaction (e.g., astreptavidin-biotin interaction). For example, particle transfer can befacilitated, e.g., by using streptavidin-coated particles, such asstreptavidin-coated nanoparticles, and a biotin-coated capture array.Alternatively, any protein-protein, protein-small-molecule, or nucleicacid-nucleic acid interaction, or any specific chemical reaction (e.g.,“click chemistry”) can be used to facilitate rapid or complete transferof nucleic acid comprising particles from the tissue sample to thecapture array.

A variety of probes can be immobilized on the particles to capturenucleic acids from the tissue sample and combined with a variety ofprobes on a capture array described herein. In some embodiments, theprobes on the particles consist essentially of capture regions tocapture the nucleic acids from the tissue sample (besides, e.g.,additional elements to immobilize the probes to the particle). In someembodiments, the probes on the particles can comprise a capture regionand a spatial address region (e.g., a partial or combinatorial spatialaddress region, or a complete spatial address region). The probe-coatedparticles described herein can be used in combination, e.g., withcapture arrays comprising capture sites having probes comprisingessentially a cleavable region, or a cleavable region and a spatialaddress region (e.g., a partial or combinatorial spatial address region,or a complete spatial address region), or a cleavable region, a spatialaddress region and a capture region (e.g., a region to capture thenucleic acids on the particles), or any other combination of regions.

In some embodiments, magnetically responsive nanoparticles are used tocapture nucleic acid (e.g., in situ synthesized cDNA) in a tissue samplefor generation of a spatially addressed library.

In some embodiments, the magnetically responsive nanoparticles cancomprise immobilized probes comprising essentially a capture region(e.g., a gene-specific or a universal capture region). In someembodiments, the probes can further comprise a SBS primer region (e.g.,a SBS3 or SBS12 region) or other universal regions, such as P5 or P7regions. In some embodiments the nanoparticles are used in combinationwith a capture array comprising capture probes comprising a cleavableregion, a spatial address region, and a capture region (e.g., agene-specific capture region). In some embodiments, the probes on thecapture array further comprise a SBS primer region (e.g., a SBS 3 orSBS12 region) or other universal regions, such as P5 or P7 regions.

FIG. 38 illustrates a flow diagram of an example of a method 3800 ofgenerating a spatially addressed sequencing library using particles,such as magnetically responsive nanoparticles, to capture nucleic acidfrom a tissue sample.

At a step 3810, cDNA is synthesized from target mRNA in a tissue sampleby in situ reverse transcription. In some embodiments, the cDNA is agene-specific (i.e., targeted) cDNA. In some embodiments, the cDNA is arandom cDNA or a cDNA representing bulk mRNA. For example, in someembodiments, a gene-specific RT primer bound to the surface of aparticle, such as a magnetically responsive nanoparticle, can be used toprime first strand cDNA synthesis in a reverse transcription reaction.

At a step 3815, first strand cDNA bound to the surface of, e.g., amagnetically responsive nanoparticle is captured onto an array. Thearray is, for example, a glass substrate that is printed with spatiallyaddressed capture probes to form an array of capture sites. Thespatially addressed capture probes can comprise, e.g., a cleavablepolylinker sequence, a spatial address sequence, and a gene-specificcapture sequence that is complementary to a sequence in the first strandcDNA. The spatially addressed capture probes can be attached to theglass substrate via the cleavable polylinker sequence. The spatialaddress sequence is typically a unique sequence for each spatial featureon the array. Each spatial feature can include a plurality of spatiallyaddressed capture probes with different gene-specific capture sequences.A magnet can be placed in proximity to the array. The reaction can beheated to an incubation temperature of about 95° C. for about 1 minuteto denature RNA:cDNA hybrids. In the proximity of the magnet, firststrand cDNA bound to the surface of a magnetically responsivenanoparticle can be anchored onto the surface of the array. First strandcDNA molecules can be captured onto the array by hybridization (e.g., atabout 60° C. for about 10 minutes) to the gene-specific capturesequences in the spatially addressed capture probes.

At a step 3820, second strand cDNA can be synthesized. For example, themagnet is removed from the proximity of the array and first strand cDNAmolecules that are not hybridized to gene-specific capture sequences inthe spatially addressed capture probes can be removed by washing. Secondstrand cDNA is synthesized in an extension reaction using the secondgene-specific capture sequence as a primer.

At a step 3825, the double-stranded cDNA can be released from thecapture array by cleavage of the cleavable polylinker sequence.

At a step 3830, double-stranded cDNA can optionally be end repaired andligated to sequencing adapters to generate a sequencing library. Inembodiments where the RT primer and the capture primer on the capturearray comprise SBS primer regions, end repair.

FIG. 39 illustrates the steps of an exemplary method 3800 of FIG. 38.Namely, a tissue section (not shown) comprises a target RNA molecule3910. Target RNA molecule 3910 can include a mutation 3915. At step3810, cDNA is synthesized in situ using a gene-specific RT primer 3920in a reverse transcription reaction. RT primer 3920 is attached to thesurface of a magnetically responsive nanoparticle 3925. A cDNA molecule3930 synthesized using RT primer 3920 is attached to magneticallyresponsive nanoparticle 3925.

At step 3815, cDNA molecules 3930 are captured onto an array thatcomprises a plurality of capture sites 3935. In this example, a singlecapture site 3935 is shown. Capture site 3935 comprises a spatiallyaddressed capture probe 3940. Spatially addressed capture probe 3940comprises a cleavable polylinker region 3945, a spatial address region3950, and a gene-specific capture region 3955 that is complementary to asequence in the first strand cDNA. Spatially addressed capture probe3940 is attached to capture site 3935 via cleavable polylinker region3945. A magnet 3960 is placed in proximity to capture site 3935. Thereaction is heated to an incubation temperature of about 95° C. forabout 1 minute to denature RNA:cDNA hybrids. In the proximity of magnet3960, first strand cDNA molecules 3930 bound to the surface ofmagnetically responsive nanoparticle 3925 are anchored onto the surfacecapture site 3935. First strand cDNA molecules 3930 are captured ontothe capture site 3935 by hybridization (e.g., at about 60° C. for about10 minutes) to gene-specific capture region 3955 in spatially addressedcapture probe 3940.

At step 3820, second strand cDNA is synthesized. For example, magnet3960 is removed from the proximity of capture site 3935 and first strandcDNA molecules 3930 that are not hybridized to gene-specific capturesequence 3955 in spatially addressed capture probe 3940 are removed bywashing. Second strand cDNA is synthesized in an extension reactionusing gene-specific capture region 3955 as a primer.

At step 3825, a double-stranded cDNA molecule 3965 that now comprisesspatial address region 3950 is released from capture site 3935 bycleavage of cleavable polylinker region 3945.

At step 3830 (not shown in FIG. 39), double-stranded cDNA molecule 3965is optionally end repaired and ligated to sequencing adapters togenerate a spatially addressed sequencing library.

In some embodiments, the RT primer and the capture primer on the capturearray can optionally and independently comprise additional regions, suchas SBS primer regions (e.g., SBS3 or SBS12 regions) or universal regions(e.g., P5 or P7 regions) that can, e.g., be incorporated into the doublestranded cDNA molecule 3965.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing amagnetically responsive nanoparticle (e.g., 3925) comprising animmobilized capture probe comprising a capture region (e.g., 3920).

In some embodiments, the capture region is a gene-specific captureregion (comprising, e.g., a TSCA sequence). In some embodiments, thecapture region is a universal capture region (comprising, e.g., a poly-Tsequence or a randomized nucleic acid sequence).

In some embodiments, the immobilized capture probe does not include aspatial addressing region.

In some embodiments, the method further comprises contacting themagnetically responsive nanoparticle with a tissue sample, such that theposition of the magnetically responsive nanoparticle on the tissuesample can be correlated with the position of a nucleic acid in thetissue sample, and allowing the nucleic acid to hybridize to the captureregion of the immobilized capture probe.

In some embodiments, the method further comprises extending the captureregion of the immobilized capture probe to form an immobilized firstcomplementary strand of the nucleic acid hybridized to the captureregion (e.g., 3930).

In some embodiments, the method further comprises contacting the tissuesample with a capture array, such that the position of a capture site onthe capture array can be correlated with a position in the tissuesample, wherein the capture array comprises a capture site (e.g., 3935)comprising a capture probe immobilized on a surface (e.g., 3940),wherein the capture probe comprises a cleavable region (e.g., 3945), aspatial address region (e.g., 3950) and a gene-specific region (e.g.,3955).

In some embodiments, the method further comprises applying a magneticfield to the capture array and tissue sample to transfer themagnetically responsive nanoparticle with the immobilized firstcomplementary strand to the capture array and allowing the immobilizedfirst complementary strand to hybridize to the capture region of thecapture probe on the capture array.

In some embodiments, the method further comprises extending the captureregion of the capture probe on the capture array to form an immobilizedsecond complementary strand of the nucleic acid (e.g., newly synthesizedstrand of 3965).

In some embodiments, the method further comprises cleaving the captureprobe on the capture array at the cleavable domain to release aspatially tagged second complementary strand from the surface of thecapture array.

In some embodiments, the method further comprises, analyzing thesequence of the released spatially tagged second complementary strand.

In some embodiments, the method further comprises correlating thesequence of the released spatially tagged second complementary stand tothe position of the nucleic acid in the tissue sample.

In some embodiments, particle (e.g., nanoparticle) associated probes canbe hybridized to a nucleic acid from a tissue sample prior toimmobilizing the probe to the nanoparticle and the probe-nucleic acidhybrid can then be immobilized to the nanoparticle. In some embodiments,a probe hybridized to a nucleic acid from the tissue sample can beextended to form a nucleic acid complementary to the nucleic acid fromthe tissue sample, and the complementary nucleic acid can then beimmobilized to the nanoparticle. In some embodiments, the probes cancomprise a linker element for linking a probe-nucleic acid hybrid or acomplementary nucleic acid to the nanoparticle.

FIGS. 40A and 40B illustrate an example of a process 4000 of using acapture probe to form a complementary nucleic acid in a tissue sampleand subsequently immobilizing the complementary nucleic acid to ananoparticle.

In a first step and referring now to FIG. 40A, a capture probe 4010 ishybridized to a nucleic acid 4015 from a tissue sample. Capture probe4010 includes a capture region 4015 and a linker element 4020. In oneexample capture region 4015 comprises a random primer sequence. Inanother example, capture region 4015 comprises a gene-specific primersequence. Linker element 4020 is an element for linking a probe-nucleicacid hybrid or complementary nucleic acid to a nanoparticle. In oneexample, nucleic acid molecule 4025 is a genomic DNA molecule. Inanother example, nucleic acid molecule 4025 is an RNA molecule. Captureprobe 4010 hybridizes to nucleic acid molecule 4025 from a tissue sampleand is extended to form a complementary nucleic acid molecule 4025 a.

In a next step and referring now to FIG. 40B, complementary nucleic acid4025 a is immobilized to a nanoparticle 4030 via linker element 4020 incapture probe 4010. In one example, nanoparticle 4030 is a magneticallyresponsive nanoparticle.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing aprimer comprising a capture region and a linker element (e.g., a biotingroup, a thiol, group, or another chemical linker).

In some embodiments, the capture region is a gene-specific captureregion (comprising, e.g., a TSCA sequence). In some embodiments, thecapture region is a universal capture region (comprising, e.g., a poly-Tsequence or a randomized nucleic acid sequence).

In some embodiments, the method further comprises contacting a tissuesample with the primer and allowing the primer to hybridize to a nucleicacid from the tissue sample.

In some embodiments, the method further comprises extending the primerto form an immobilized first complementary strand of the nucleic acidhybridized to the primer.

In some embodiments, the method further comprises contacting the tissuesample with a magnetically responsive nanoparticle (e.g., a streptavidincoated magnetically responsive nanoparticle), such that the position ofthe magnetically responsive nanoparticle on the tissue sample can becorrelated with the position of the extended primer in the tissuesample, and immobilizing the extended primer comprising the firstimmobilized strand of the nucleic acid to the magnetically responsivenanoparticle at the linker element.

In some embodiments, the method further comprises contacting the tissuesample with a capture array such that the position of a capture site onthe capture array can be correlated with a position in the tissuesample, wherein the capture array comprises a capture site comprising acapture probe immobilized on a surface, wherein the capture probecomprises a cleavable region, a spatial address region and agene-specific region.

In some embodiments, the method further comprises applying a magneticfield to the capture array and the tissue sample to transfer themagnetically responsive nanoparticle with the immobilized firstcomplementary strand to the capture array and allowing the immobilizedfirst complementary strand to hybridize to the gene-specific region ofthe capture probe on the capture array.

In some embodiments, the method further comprises extending thegene-specific region of the capture probe on the capture array to forman immobilized second complementary strand of the nucleic acid.

In some embodiments, the method further comprises cleaving the captureprobe on the capture array to release a spatially tagged secondcomplementary strand from the surface of the capture array.

In some embodiments, the method further comprises, analyzing thesequence of the released spatially tagged second complementary strand.

In some embodiments, the method further comprises correlating thesequence of the released spatially tagged second complementary stand tothe position of the nucleic acid in the tissue sample

In some embodiments, a particle (e.g., a magnetically responsivenanoparticle) can comprise an immobilized probe comprising a captureregion (e.g., a gene-specific or a universal capture region) and a firstpartial address region. In some embodiments, the particle associatedprobe can further comprise a SBS primer region (e.g., a SBS3 or SBS12region) or another universal region, such as a P5 or P7 region. In someembodiments the particles can be used in combination with a capturearray comprising a capture probe comprising a cleavable region, a secondspatial address region, and a capture region (e.g., a gene-specificcapture region). In some embodiments, the probe on the capture array canfurther comprise an SBS primer region (e.g., an SBS 3 or SBS12 region)or another universal region, such as a P5 or P7 region.

FIGS. 41A and 41B illustrate schematic diagrams of an example of aparticle-associated capture probe comprising a first partial spatialaddress region and a second array capture probe comprising a secondpartial address region, respectively, for spatial detection and analysisof nucleic acids in a tissue sample. Referring now to FIG. 41A, aparticle-associated capture probe 4110 comprises a capture region 4115(e.g., a gene-specific or a universal capture region) and a firstpartial address region 4120 immobilized on a particle 4125. In oneexample, particle 4125 is a magnetically responsive nanoparticle.Capture probe 4110 hybridizes to a nucleic acid molecule 4130 from atissue section and is extended (indicated by the arrow) to form anucleic acid complementary to nucleic acid molecule 4130. Thecomplementary nucleic acid comprises first partial address region 4120and particle 4125. First partial address region 4120 identifies theposition of a capture site along a first dimension of a capture array.In some embodiments, a magnetic field is used to facilitate transfer ofthe complementary nucleic acid molecule comprising particle 4125 onto anarray comprising an array capture probe.

Referring now to FIG. 41B, an array capture probe 4135 comprises acapture region 4140 (e.g., a gene-specific capture region), a secondpartial address region 4145, and SBS region 4150, and a cleavable region4155. Array capture probe 4135 can be immobilized on the surface of acapture array (not shown) via cleavable region 4155. Array capture probe4135 on a capture array (not shown) can be contacted with a tissuesample comprising nucleic acid molecules labeled with first partialaddress region 4120 of FIG. 41A such that the position of array captureprobe 4135 can be correlated with a position in the tissue sample.Second partial address region 4145 identifies the position of a capturesite along a second dimension of a capture array.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing amagnetically responsive nanoparticle comprising an immobilized captureprobe comprising a capture region and a first partial spatial addressregion.

In some embodiments, the capture region is a gene-specific captureregion (comprising, e.g., a TSCA sequence). In some embodiments, thecapture region is a universal capture region (comprising, e.g., a poly-Tsequence or a randomized nucleic acid sequence).

In some embodiments, the first partial spatial address region identifiesthe position of a capture site along a first dimension of a capturearray.

In some embodiments, the method further comprises contacting themagnetically responsive nanoparticle with a tissue sample, such that theposition of the magnetically responsive nanoparticle on the tissuesample can be correlated with the position of a nucleic acid in thetissue sample, and allowing the nucleic acid to hybridize to the captureregion of the immobilized capture probe.

In some embodiments, the method further comprises extending the captureregion of the immobilized capture probe to form an immobilized firstcomplementary strand of the nucleic acid hybridized to the captureregion, wherein the immobilized first complementary strand comprises thefirst partial address region.

In some embodiments, the method further comprises contacting the tissuesample with a capture array such that the position of a capture site onthe capture array can be correlated with a position in the tissuesample, wherein the capture array comprises a capture site comprising acapture probe immobilized on a surface, wherein the capture probecomprises a cleavable region, a second partial address region and agene-specific region.

In some embodiments, the second partial spatial address regionidentifies the position of a capture site along a second dimension of acapture array.

In some embodiments, the method further comprises applying a magneticfield to the capture array and tissue sample to transfer themagnetically responsive nanoparticle with the immobilized firstcomplementary strand to the capture site of a capture array and allowingthe immobilized first complementary strand to hybridize to the captureregion of the capture probe on the capture site of the capture array.

In some embodiments, the method further comprises extending the captureregion of the capture probe on the capture site to form an immobilizedsecond complementary strand of the nucleic acid, wherein the secondcomplementary strand of the nucleic acid comprises the first and secondpartial spatial address regions.

In some embodiment, the combination of the first and second partialspatial address region defines the position of the capture site on thecapture array.

In some embodiments, the method further comprises cleaving the captureprobe on the capture array at the cleavable domain to release aspatially tagged second complementary strand from the surface of thecapture array.

In some embodiments, the method further comprises, analyzing thesequence of the released spatially tagged second complementary strand.

In some embodiments, the method further comprises correlating thesequence of the released spatially tagged second complementary stand tothe position of the nucleic acid in the tissue sample.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing amagnetically responsive nanoparticle comprising an immobilized captureprobe comprising a capture region.

In some embodiments, the immobilized capture probe does not comprise aspatial address region.

In some embodiments, the capture region is a gene-specific captureregion (comprising, e.g., a TSCA sequence). In some embodiments, thecapture region is a universal capture region (comprising, e.g., a poly-Tsequence or a randomized nucleic acid sequence).

In some embodiments, the method further comprises contacting themagnetically responsive nanoparticle with a tissue sample, such that theposition of the magnetically responsive nanoparticle on the tissuesample can be correlated with the position of a nucleic acid in thetissue sample, and allowing the nucleic acid to hybridize to the captureregion of the immobilized capture probe.

In some embodiments, the method further comprises extending the captureregion of the immobilized capture probe to form an immobilized firstcomplementary strand of the nucleic acid hybridized to the captureregion.

In some embodiments, the immobilized first complementary strand does notcomprise a spatial address region.

In some embodiments, the method further comprises contacting the tissuesample with a capture array such that the position of a capture site onthe capture array can be correlated with a position in the tissuesample, wherein the capture array comprises a capture site comprising aprobe immobilized on a surface, wherein the probe comprises a spatialaddress region.

In some embodiments, the probe does not comprise a capture region.

In some embodiments, the method further comprises applying a magneticfield to the capture array and tissue sample to transfer themagnetically responsive nanoparticle with the immobilized firstcomplementary strand to the capture site of a capture array and ligatingthe immobilized first complementary strand to the spatial address regionof the probe on the capture array to immobilize the first complementarystrand on both ends on the capture array and on the magneticallyresponsive nanoparticle.

FIGS. 42A and 42B illustrate schematic diagrams of an example of aparticle-associated capture probe and a second array capture probecomprising a spatial address region, respectively, for spatial detectionand analysis of nucleic acids in a tissue sample. Referring now to FIG.42A, a particle-associated capture probe 4210 comprises a capture region4215 (e.g., a gene-specific or a universal capture region) immobilizedon a magnetically responsive nanoparticle 4220. Capture probe 4210hybridizes to a nucleic acid molecule 4225 from a tissue section and isextended (indicated by the arrow) to form a nucleic acid complementaryto nucleic acid molecule 4225. The complementary nucleic acid comprisesmagnetically responsive nanoparticle 4220. In some embodiments, amagnetic field is used to facilitate the transfer of the complementarynucleic acid molecule comprising magnetically responsive nanoparticle4220 onto an array comprising an array capture probe.

Referring now to FIG. 42B, an array capture probe 4230 comprises aspatial address region 4235 a cleavable region 4240. Array capture probe4230 can be immobilized on the surface of a capture array (not shown)via cleavable region 4240. Array capture probe 4230 on a capture arraysurface (not shown) can be contacted with a tissue sample comprising thecomplementary nucleic acid molecules tagged with magnetically responsivenanoparticle 4220 of FIG. 42A such that the position of array captureprobe 4230 can be correlated with a position in the tissue sample. Thecomplementary nucleic acids comprising magnetically responsivenanoparticle 4220 can be captured on to capture probe 4230 by ligationto spatial address region 4235. Spatial address region 4240 identifiesthe position of a capture site on the array.

In some embodiments, the first complementary strand comprises a spatialaddress region when immobilized on both ends on the capture array and onthe magnetically responsive nanoparticle.

In some embodiments, the immobilized capture probe on the magneticallyresponsive nanoparticle and the spatial address region optionally eachfurther comprise a primer binding region (e.g., a SBS primer bindingregion, such as a SBS3 or SBS12 region). In some embodiments, the methodfurther comprises synthesizing a second complementary strand using aprimer pair complementary to the primer binding regions in the firstcomplementary strand, wherein the second complementary strand comprisesthe spatial address region, and releasing the second complementarystrand from the surface of the capture array. In some embodiments, themethod further comprises analyzing the sequence of the released secondcomplementary strand and correlating the sequence of the released secondcomplementary stand to the position of the nucleic acid in the tissuesample.

In some embodiments, the immobilized capture probe on the magneticallyresponsive nanoparticle and the spatial address region optionally eachfurther comprise a cleavable region (e.g., the same cleavable region ordifferent cleavable regions). In some embodiments, the method furthercomprises releasing the immobilized first complementary strand bycleaving the cleavable regions in the immobilized first complementarystrand. In some embodiments, the method further comprises analyzing thesequence of the released first complementary strand and correlating thesequence of the released second complementary stand to the position ofthe nucleic acid in the tissue sample.

In some embodiments, a particle (e.g., a magnetically responsivenanoparticle) can comprise an immobilized probe comprising a captureregion (e.g., a gene-specific or a universal capture region), a firstprimer binding region (e.g., an SBS primer region, such as an SBS3 orSBS12 region) and a spatial address region. In some embodiments, theparticle associated probe can further comprise another universal region,such as a P5 or P7 region. In some embodiments the particles can be usedin combination with a capture array comprising a capture probecomprising essentially a second primer binding region (e.g., an SBSprimer region, such as an SBS 3 or SBS12 region) and, optionally anotheruniversal region, such as a P5 or P7 region.

FIGS. 43A and 43B illustrate schematic diagrams of an example of aparticle-associated capture probe and a second array capture probe,respectively, for spatial detection and analysis of nucleic acids from atissue sample. Referring now to FIG. 43A, a particle-associated captureprobe 4310 comprises a capture region 4315 (e.g., a gene-specific or auniversal capture region), a spatial address region 4320, and a firstprimer binding site 4325 (e.g., an SBS primer region SBS3) immobilizedon a magnetically responsive nanoparticle 4330. Capture probe 4310 canbe contacted with a tissue sample (not shown), such that the position ofcapture probe 4310 on the tissue sample can be correlated with theposition of a nucleic acid molecule 4335 in the tissue sample. Captureprobe 4310 hybridizes to nucleic acid molecule 4335 from the tissuesection and is extended (indicated by the arrow) to form a nucleic acidcomplementary to nucleic acid molecule 4335. The complementary nucleicacid (not shown) comprises spatial address region 4320, first primerbinding site 4325, and magnetically responsive nanoparticle 4330. Insome embodiments, a magnetic field generated by a magnet (not shown) isused to facilitate the transfer of the complementary nucleic acidmolecule comprising magnetically responsive nanoparticle 4330 onto anarray comprising an array capture probe.

Referring now to FIG. 43B, an array capture probe 4340 comprises asecond primer binding site 4325 a (e.g., an SBS primer region SBS12)that is different from first primer binding site 4325 of FIG. 43A. Arraycapture probe 4340 can be immobilized on the surface of a capture array(not shown). Array capture probe 4340 on a capture array surface (notshown) can be contacted with a tissue sample comprising thecomplementary nucleic acid molecules tagged with magnetically responsivenanoparticle 4330 of FIG. 43A. The complementary nucleic acid comprisingmagnetically responsive nanoparticle 4330 can be captured onto captureprobe 4340 by ligation to second primer binding site 4325 a.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing amagnetically responsive nanoparticle comprising an immobilized captureprobe comprising a capture region, a first primer binding region and aspatial address region.

In some embodiments, the capture region is a gene-specific captureregion (comprising, e.g., a TSCA sequence). In some embodiments, thecapture region is a universal capture region (comprising, e.g., a poly-Tsequence or a randomized nucleic acid sequence).

In some embodiments, the method further comprises contacting themagnetically responsive nanoparticle with a tissue sample, such that theposition of the magnetically responsive nanoparticle on the tissuesample can be correlated with the position of a nucleic acid in thetissue sample, and allowing the nucleic acid to hybridize to the captureregion of the immobilized capture probe.

In some embodiments, the method further comprises extending the captureregion of the immobilized capture probe to form an immobilized firstcomplementary strand of the nucleic acid hybridized to the captureregion, wherein the immobilized first complementary strand comprises thespatial address region.

In some embodiments, the method further comprises contacting the tissuesample with a capture array such that the position of a capture site onthe capture array can be correlated with a position in the tissuesample, wherein the capture array comprises a capture site comprising acapture probe immobilized on a surface, wherein the capture probecomprises essentially a second primer binding region (e.g., an SBSprimer region, such as an SBS3 or SBS12 region).

In some embodiments, the method further comprises applying a magneticfield to the capture array and tissue sample to transfer themagnetically responsive nanoparticle with the immobilized firstcomplementary strand to the capture site of a capture array and ligatingthe immobilized first complementary strand to the capture probe on thecapture site of the capture array to immobilize the first complementarystrand at both ends.

In some embodiments, the method further comprises synthesizing a secondcomplementary strand using a primer complementary to the first primerbinding sequence of the first capture probe and releasing the secondcomplementary strand from the surface of the capture array.

In some embodiments, the method further comprises analyzing the sequenceof the released second complementary strand and correlating the sequenceof the released second complementary stand to the position of thenucleic acid in the tissue sample.

FIG. 44 illustrates a perspective view of a magnetic-based transfersystem 4400 that is configured for spatial detection and analysis ofnucleic acid in a tissue sample. Magnetic-based transfer system 4400includes capture array 4410. Capture array 4410 includes a solid support4415. In one example, solid support 4415 is a planar glass substrate.Printed on the surface of solid support 4415 are a plurality of distinctcapture sites (“spatial features”) 4420, which are regions containingspatially addressed oligonucleotides. An example of a single capturesite 4420 is described in more detail with reference to FIG. 45.Overlaid on capture array 4410 is a sample substrate 4425. In oneexample, sample substrate 4425 is a glass slide. A tissue sample 4430 ismounted on the surface of sample substrate 4425 that is facing capturesites 4420 on capture array 4410. In one example, tissue section 4430 isan FFPE tissue section. Nucleic acid (not shown) in tissue sample 4430is tagged with a magnetically responsive nanoparticle. Positioned belowand in proximity to capture array 4410 is a magnet 4435. Magnet 4435 maybe a permanent magnet or an electromagnet. In one example, magnet 4435is a movable magnet. Namely, magnet 4435 may be moved in proximity to oraway from capture array 4410. A magnetic field generated by magnet 4435is used to attract the nanoparticle-tagged nucleic acid molecules (notshown) from tissue section 4430 onto capture sites 4420 on capture array4410. The magnetic field generated by magnet 4435 is configured suchthat transfer of nucleic acids from the tissue sample and loss ofnucleic acids between capture sites 4420 is eliminated or substantiallyreduced.

In another embodiment (not shown), capture sites 4420 are microwells insolid support 4415. Printed on the bottom surface of each microwell arethe spatially addressed oligonucleotides. In the presence of a magneticfield, the microwells function to trap the nanoparticle-tagged nucleicacid and eliminate or substantially reduce aggregation of themagnetically responsive nanoparticles.

FIG. 45 illustrates a side view of one capture site 4420 on capturearray 4410, wherein the one capture site 4420 includes a plurality ofcapture probes. In one example, a plurality of capture probes 4510includes an SBS primer sequence 4515 (e.g., SBS3) and a spatial addresssequence 4520. In another example (not shown), capture 4510 includes aP7 sequence, an SBS primer sequence (e.g., SBS12), and a spatial addresssequence as described in more detail with reference to FIG. 49A. Eachcapture probe 4510 immobilized at a single capture site 4420 includesthe same unique spatial address sequence 4520 (e.g., spatial addresssequence 4520 a). For example, in FIG. 45, both capture probe 4510 a andcapture probe 4510 b have the same unique spatial address sequence 4520a. Other capture sites 4420 (not shown) each include their own uniquespatial address sequence 4520 (e.g., spatial address sequences 4520 b,4520 c, 4520 d, 4520 e, and so on). Accordingly, capture probes 4510immobilized at other capture sites 4420 each include their own uniquespatial address sequence 4520. SBS primer sequence 4515 is used insubsequent processing steps for library preparation.

FIG. 46 illustrates a flow diagram of an example of a method 4600 oftransferring cDNA from a tissue sample to a capture array for generationof a spatially addressed sequencing library using magnetic-basedtransfer system 4100 of FIG. 44 and FIG. 45. Method 4600 includes, butis not limited to, the following steps.

At a step 4610, first strand cDNA is synthesized in situ from RNA in atissue sample in a reverse transcription (RT) reaction. For example, anRT primer bound to the surface of a magnetically responsive nanoparticleis used to prime first strand cDNA from mRNA in tissue sample 4430mounted on sample substrate 4425. In one example, the RT primer includesa gene-specific primer sequence and an SBS primer sequence (e.g., SBS3).In another example, the RT primer includes random primer sequences andan SBS primer sequence (e.g., SBS3). Because the RT primer is bound tothe surface of a magnetically responsive nanoparticle, first strand cDNAis tagged with the magnetically responsive nanoparticle.

At a step 4615, first strand cDNA is transferred onto a spatiallyaddressed capture array using a magnetic field. For example, samplesubstrate 4425 with tissue sample 4430 thereon is placed atop capturearray 4410. Magnet 4435 is positioned in close proximity to capturearray 4410. The magnetic field generated by magnet 4435 is used toattract the nanoparticle-tagged first strand cDNA from tissue section4430 onto capture sites 4420 on capture array 4410. Accordingly, firststrand cDNA is immobilized at capture sites 4420 by magnet 4435.

At a step 4620, first strand cDNA is covalently linked to capture probes4510 by single-strand ligation of the 3′ end of the cDNA to spatialaddress sequence 4520.

At a step 4625, magnet 4435 is moved away from capture array 4410 suchthat capture array 4410 is no longer within the magnetic field of magnet4435. As the magnetic field at capture array 4410 diminishes, the 5′ endof the first strand cDNA molecule is released from capture site 4420.The first strand cDNA is now anchored at capture site 4420 via captureprobe 4510.

At a step 4630, second strand cDNA is synthesized using a primer that iscomplementary to SBS primer sequence 4515 on capture probes 4510.

At a step 4635, the second strand cDNA is released from capture site4420. In one example, the second strand cDNA is released from capturesite 4420 using a heat denaturation protocol. In another example, thesecond strand cDNA is released using a chemical (e.g., NaOH)denaturation protocol.

At a step 4640, the second strand cDNA is amplified to generate asequencing library.

FIGS. 47A, 47B, and 47C show pictorially the steps of method 4600 ofFIG. 46. Namely, a tissue section (not shown) includes an RNA molecule4710. RNA molecule 4710 may include a mutation 4715. At step 4610, firststrand cDNA is synthesized in situ using an RT primer 4720. RT primer4720 includes a random primer sequence 4725 and an SBS primer sequence4730 (e.g., SBS3). RT primer 4720 is bound to the surface of amagnetically responsive nanoparticle 4735. A first strand cDNA molecule4740 synthesized using RT primer 4720 includes SBS primer sequence 4730and magnetically responsive nanoparticle 4735.

At step 4615, first strand cDNA molecules 4740 are transferred from thetissue section (not shown) onto capture site 4420. Magnet 4435 ispositioned in close proximity to capture array 4410. The magnetic fieldgenerated by magnet 4435 is used to attract nanoparticle-tagged firststrand cDNA molecules 4740 from the tissue section (not shown) ontocapture site 4420. Accordingly, first strand cDNA molecules 4740 areimmobilized at capture sites 4420 by magnet 4435.

At step 4620, first strand cDNA molecules 4740 are covalently linked tocapture probes 4510 by single-strand ligation of cDNA molecules 4740 tospatial address sequences 4520.

At step 4625, magnet 4435 is moved away from capture array 4410 suchthat capture array 4410 is no longer within the magnetic field of magnet4435. As the magnetic field at capture array 4410 diminishes, the 5′ endof first strand cDNA molecules 4740 are released from capture site 4420.First strand cDNA molecules 4740 are now anchored at capture site 4420via capture probe 4510.

At step 4630, a second strand cDNA molecule 4745 is synthesized using aprimer 4515 a that is complementary to SBS primer sequence 4515 oncapture probes 4510.

At step 4635, second strand cDNA molecule 4745 is released from capturesite 4420. Second strand cDNA molecule 4745 includes SBS primer sequence4730 (e.g., SBS3), mutation 4715, spatial address sequence 4520, and SBSprimer sequence 4515 (e.g., SBS12).

At step 4640, second strand cDNA molecule 4745 is amplified using afirst SBS primer 4750 and a second primer 4755 to generate a sequencinglibrary. First SBS primer 4750 includes an SBS complementary sequence4730 a that is complementary to SBS primer sequence 4730 and a P5sequence 4760. Second SBS primer 4755 includes an SBS complementarysequence 4515 a that is complementary to SBS primer sequence 4515 and aP7 sequence 4765. A library amplicon 4770 synthesized using SBS primers4750 and 4755 includes P5 sequence 4760, SBS primer sequence 4730 (e.g.,SBS3), mutation 4715, spatial address sequence 4520, SBS primer sequence4515 (e.g., SBS12), and P7 sequence 4765.

In another embodiment, RNA in a tissue sample (e.g., an FFPE tissuesample) is tagged in situ with magnetically responsive nanoparticles andsubsequently transferred to a capture array for generation of aspatially addressed cDNA library.

FIG. 48 illustrates a flow diagram of an example of a method 4800 oftransferring RNA from a tissue sample to a capture array for generationof a spatially addressed sequencing library using magnetic-basedtransfer system 4400 of FIG. 44. Method 4800 includes, but is notlimited to, the following steps.

At a step 4810, RNA in a tissue sample is tagged with magneticallyresponsive nanoparticles. For example, an SBS primer oligonucleotidebound to the surface of a magnetically responsive nanoparticle isligated to the RNA in a tissue sample. The 3′ end of the RNA moleculesare then modified (i.e., blocked) to prevent unwanted ligation in asubsequent processing step.

At a step 4815, the tagged RNA is transferred onto capture site 4420using a magnetic field as described above for cDNA in step 4615 ofmethod 4600 of FIG. 46.

At a step 4820, first strand cDNA is synthesized from the transferredRNA in a RT reaction. For example, an RT primer that includes agene-specific primer sequence and a ligation oligonucleotide is used toprime the first strand cDNA. In another example, an RT primer thatincludes random primer sequences and a ligation oligonucleotide is usedto prime the first strand cDNA.

At a step 4825, the first strand cDNA is covalently linked to captureprobes on capture site 4420 by single-strand ligation. The captureprobes are described in more detail with reference to FIG. 49A.

At a step 4830, the RNA template used to generate the cDNA is releasedfrom capture site 4420. For example, magnet 4435 is moved away fromcapture array 4410. Thereby diminishing the magnetic field at capturesite 4420 and releasing the magnetically responsive nanoparticle fromthe surface capture sites 4420. RNA:cDNA duplexes are dissociated usinga heat treatment protocol. The first strand cDNA is anchored at capturesite 4420 via ligation to the capture probes.

At a step 4835, second strand cDNA is synthesized in an extensionreaction. For example, a primer that includes a sequence that iscomplementary to the SBS primer sequence in the cDNA and a P5 sequenceis used to primer the second strand cDNA synthesis.

At a step 4840, cDNA library molecules are released from capture site4420 by denaturation.

FIGS. 49A, 49B, and 49C show pictorially the steps of method 4800 ofFIG. 48. Namely, a tissue section (not shown) includes an RNA molecule4910. RNA molecule 4910 may include a mutation 4915. At step 4810, anSBS primer oligonucleotide 4920 bound to the surface of a magneticallyresponsive nanoparticle 4925 is ligated in situ to RNA molecule 4910.The 3′ end of RNA molecule 4910 is then modified (i.e., blocked) toprevent unwanted ligation in a subsequent processing step.

At step 4815, RNA molecule 4910 with magnetically responsivenanoparticle 4925 thereon is transferred from the tissue section (notshown) onto capture site 4420 using a magnetic field as described abovefor cDNA in step 4615 of method 4600 of FIG. 46. In this example,capture site 4420 includes a capture probe 4935. Capture probe 4935includes a P7 sequence 4940, an SBS primer sequence 4945 (e.g., SBS12),and a spatial address sequence 4950.

At step 4820, cDNA is synthesized from RNA molecule 4910 in a RTreaction. For example, an RT primer 4955 that includes a gene-specificprimer sequence 4960 and a ligation oligonucleotide 4965 is used toprime first strand cDNA.

At step 4825, a cDNA molecule 4970 that includes SBS primer sequence4920 (e.g., SBS3) and ligation oligonucleotide 4965 is covalently linkedto capture probe 4935 by single-strand ligation of ligationoligonucleotide 4965 to spatial address sequence 4950.

At step 4830, RNA molecule 4910 is released from capture site 4420. Forexample, magnet 4435 is moved away from capture site 4420. Therebydiminishing the magnetic field at capture site 4420 and releasingmagnetically responsive nanoparticle 4925 bound to RNA molecule 4910from the surface capture site 4420. The RNA:cDNA duplex (i.e., RNAmolecule 4910:cDNA molecule 4970 duplex) is then dissociated using aheat treatment protocol. cDNA molecule 4970 is now anchored at capturesite 4420 by capture probe 4935.

At step 4835, cDNA molecule 4970 is copied in an extension reaction. Forexample, a primer 4975 that includes a sequence 4920 a that iscomplementary to SBS primer sequence 4920 (e.g., SBS3) and a P5 sequence4980 is used to primer second strand cDNA synthesis. cDNA molecule 4970now includes P7 sequence 4940, SBS primer sequence 4945 (e.g., SBS12),spatial address sequence 4950, ligation oligonucleotide 4965, mutation4915, SBS primer sequence 4920 (e.g., SBS3), and P5 sequence 4980.

At step 4840, a cDNA molecule 4970 is released from capture site 4420 bydenaturation (e.g., heat or chemical denaturation). cDNA molecule 4970is now ready for sequencing.

4.7 Spatial Tissue Profiling Based on DNA

The disclosed techniques provide methods of spatial detection andanalysis (e.g., mutational analysis or single nucleotide variation (SNV)detection) of genomic DNA in a tissue sample. In one example, the tissuesample is an FFPE tissue sample. Spatial detection and analysis of theDNA in a tissue sample (e.g., an FFPE sample) has several advantagescompared to spatial detection and analysis of RNA in a tissue sample:(1) DNA is more stable than RNA; (2) DNA fragments in an FFPE tissuesample are longer (e.g., 300-400 bp) compared to RNA fragments in anFFPE tissue sample (e.g., 100-200 bp); (3) RNA molecules expressed at arelatively low level may be undetectable; and (4) changes in tumorsuppressor genes are detected in DNA while they are not detected in RNA.

A disadvantage of using DNA for spatial tissue profiling is that formost genes there may be only 2 copies of a gene per cell. The methodsdisclosed herein include an initial in situ whole genomepre-amplification step that is used to increase gene copy number priorperforming other biochemical process steps.

FIG. 50 illustrates a flow diagram of an example of a method 5000 ofprofiling genomic DNA in a tissue sample. In one example, method 5000 isused for profiling SNVs of interest in genomic DNA. Method 5000includes, but is not limited to, the following steps.

At a step 5010, a glass substrate is printed with spatially addressedPCR primers to form an array of spatial features. In one example, thespatially addressed PCR primers are printed on a 2 cm×2 cm coverslip toform an array of spatial features that are 100 μm in diameter on a pitchof 35 μm. In another example, the spatially addressed PCR primers areprinted in microwells fabricated on the surface of a glass slide. Thespatially addressed PCR primers are printed on a coverslip or glassslide using, for example, commercially available printing technologies.The spatially addressed PCR primers include a random primer sequence, aspatial address sequence, an SBS primer sequence and a biotin label asdescribed in more detail with reference to FIG. 51. The spatiallyaddressed PCR primers may also include a modification at the 5′ end ofthe molecule for reversible attachment to the coverslip. In one example,the spatially addressed PCR primers may include a 5′ disulfidemodification as described for spatially addressed capture probe 3100 ofprocess 3200 of FIGS. 32A, 32B, and 32C. In another example, thespatially addressed PCR primers may include a 5′ photocleavable linkeras described for spatially addressed capture probe 3100 of process 3300of FIGS. 33A, 33B, and 33C.

At a step 5015, a PCR master mix solution is dispensed onto the surfaceof a semi-permeabilized FFPE tissue section mounted on a glass slide.The PCR master mix solution includes, for example, dNTPs, DNApolymerase, MgCl₂, and reaction buffers.

At a step 5020, the glass substrate with spatially addressed PCR primersthereon is placed atop the semi-permeabilized FFPE tissue section, suchthat the surface of the glass substrate with the spatially addressed PCRprimers thereon is in contact with the tissue section. The spatiallyaddressed PCR primers are released from the surface of the glasssubstrate into the cellular space of the tissue section.

At a step 5025, genomic DNA is amplified by in situ isothermalamplification. In one example, the amplification reaction is arecombinase polymerase amplification (RPA) reaction. Table 1 below showsother examples of isothermal DNA amplification methods that may be usedto amplify the genomic DNA. In another example, a conventional PCR-basedwhole genome amplification reaction is used to amplify the genomic DNA.In one example, the conventional PCR-based method is improved primerextension pre-amplification PCR (iPEP PCR). In another example, theconventional PCR-based method is degenerate oligonucleotide-primed PCR(DOP-PCR; e.g., Rubicon Picoplex kit).

TABLE 1 Summary of isothermal nucleic acid amplification methodsAmplification Reaction Method* time volume Target Detection limit LAMPwithin 1 h 25 μL Hepatitis B virus 50 copies/25 μL (HBV) DNA within 15min 10 μL Prostate-specific 23 fg/μL antigen gene within 1 h  5 μLPseudorabies virus 10 fg (PRV) DNA within 1 h — λ DNA two molecule 1 h35 min 35 μL E. coli genomic 24 colony forming DNA units (CFU)/mL 48CRU/mL HDA   2 h 150 μL N. gonorrhoeae 1 ng genomic DNA Methicillinresistant 250 pg S. aureus genomic DNA 0.5 h ~5 μL/192 nL BNI-1 fragmentof 0.01 ng/μL SARS cDNA 0.5 h 25 μL E. coli genomic 10 CFU DNA RCAwithin 65 min 10 μL Genomic DNA for 25 ng V. cholera   4 h  2 pLpIVEX2.2EM-lacZ 0.07 pg/μL plasmid 2.5 h pL Human-malaria- less than onecausing Plasmodium parasite/μL parasites MDA 10-16 h  60 nL E. coligenomic — DNA RPA within 20 min 10 μL mecA gene of less than 10 copiesStaphylococcus aureus   1 h  9 nL Methicillin-resistant 300 copies/mLStaphylococcus aureus genomic DNA *LAMP = loop-mediated isothermalamplification; HDA = helicase dependent amplification; MDA = multipledisplacement amplification; RCA = rolling circle amplification; RPA =recombinase polymerase amplification

At a step 5030, the semi-permeabilized tissue sample with the amplifiedgenomic DNA therein is removed from the surface of the glass slide andcollected in a collection tube. In one example, the semi-permeabilizedtissue sample with the amplified genomic DNA therein is removed from theglass slide by scraping into an Eppendorf tube. Because of the biotinlabel on the PCR primer used in the amplification reaction, theamplified DNA is biotinylated. The amplified biotinylated DNA ispurified using a streptavidin bead-based purification protocol.

At a step 5035, residual single-stranded PCR primers are removed bydigestion using a 3′ to 5′ exonuclease.

At a step 5040, the DNA is amplified in a multiplex PCR reactiontargeting SNVs of interest. For example, a forward primer includes agene-specific sequence that targets an SNV of interest, an SBS primersequence (e.g., SBS3), and a P5 sequence. A reverse primer includesSBS12 complementary sequences and a P7 sequence. In another example, aTSCA-like approach is used to target DNA regions of interest.

At a step 5045, the PCR product is sequenced.

FIG. 51 illustrates a diagram of a spatially addressed PCR primer 5100for pre-amplification and spatial indexing of whole genomic DNA.Spatially addressed PCR primer 5100 includes a random primer sequence5110, a spatial address sequence 5115, and an SBS primer sequence 5120(e.g., SBS12). Random primer sequence 5110 is a 9 bp sequence that isused to amplify genomic DNA in a whole genome pre-amplificationreaction. Spatial address sequence 5115 is a unique sequence for eachcapture site (spatial feature) on an array. SBS primer sequence 5120includes biotin label 5125. Biotin label 5125 is used to purify theamplified DNA in subsequent processing steps.

FIGS. 52A and 52B show pictorially the steps of method 5000 of FIG. 50.At step 5010, a glass substrate 5210 is printed with spatially addressedPCR primers (not shown) to form an array of spatial features 5215. Inthis example, glass substrate 5210 is a 2 cm×2 cm coverslip with spatialfeatures that are 100 μm in diameter. The spatially addressed PCRprimers are, for example, spatially addressed PCR primer 5100 of FIG.51.

At step 5015, a semi-permeabilized tissue section 5220 mounted on aglass slide 5225 is overlaid with a PCR master mix solution 5230.Semi-permeabilized tissue sample 5220 includes a cell 5235. Cell 5235includes a genomic DNA molecule 5240. Genomic DNA molecule 5240 mayinclude a single nucleotide variation (SNV) 5245.

At step 5020, glass substrate 5210 is placed atop tissue section 5220and PCR master mix solution 5230 such that the surface glass substrate5210 with spatially addressed PCR primers 5100 thereon (not shown) is incontact with tissue section 5220. PCR primers 5100 (not shown) arereleased from the surface of glass substrate 5210 into the cellularspace of tissue section 5220.

At step 5025, genomic DNA is amplified by in situ isothermalamplification using PCR primers 5100.

At step 5030, tissue section 5220 with amplified DNA 5260 therein isremoved from the surface of glass slide 5225 and collected in acollection tube 5255. In one example, collection tube 5255 is anEppendorf tube. Because of biotin label 5125 on PCR primer 5100,amplified DNA 5260 is biotinylated.

At step 5035 (not shown in FIG. 52), amplified DNA 5260 is purifiedusing a streptavidin bead-based purification protocol.

At step 5040 (not shown in FIG. 52), residual single-stranded PCRprimers 5100 are removed by digestion using a 3′ to 5′ exonuclease.

At step 5045, DNA molecules 5260 are amplified in a target-specificmultiplex PCR reaction. For example, a forward primer 5265 includes agene-specific sequence 5270 that targets SNV 5240, an SBS primersequence 5275 (e.g., SBS3), and a P5 sequence 5280. A reverse primer5285 includes a SBS12 complementary sequence 5120 a and a P7 sequence5290. A library amplicon 5295 synthesized using forward primer 5265 andreverse primer 5285 includes P5 sequence 5285, SBS primer sequence 5275,SNV 5240, spatial address sequence 5115, SBS primer sequence 5120, andP7 sequence 5290.

4.8 Spatial Compartmentalization

To limit the diffusion of spatially addressed oligonucleotides (andother reaction components or products) on a tissue section and maintainspatial resolution, compartmentalization of biochemical reactions in“microwell reactors” or “microreactors” may be used. Spatialcompartmentalization can be combined with any of the biochemistrytechniques described herein for characterization of transcriptomesand/or genomic variation in tissues while preserving spatial informationrelated to the origin of target nucleic acids in the tissue.

FIG. 53 illustrates a perspective view of an example of a microwellreactor overlay 5300. Microwell reactor overlay 5300 includes a glassslide 5310. Mounted atop glass slide 5310 is a tissue section 5315. Inone example, tissue section 5315 is an FFPE tissue section. A reactionfluid 5320 is dispensed onto glass slide 5310 such that tissue section5315 is covered by reaction fluid 5320. In one example, reaction fluid5320 is a PCR master mix solution used in an amplification reaction. Inanother example, reaction fluid 5320 is a reverse transcription mixsolution. A microwell substrate 5325 is placed atop reaction fluid 5320and tissue section 5315 on glass slide 5320. Microwell substrate 5325includes a plurality of microwells 5330. Printed on the bottom surfaceof each microwell 5330 is a plurality of spatially addressedoligonucleotides (not shown). Microwells 5330 function ascompartmentalized reaction chambers for performing a biochemicalreaction (e.g., PCR amplification). Microwells 5330 are described inmore detail with reference to FIG. 54.

FIG. 54 illustrates a perspective view of a single microwell 5330 ofmicrowell reactor overlay 5300 of FIG. 53. In this example, microwell5330 has a diameter of about 200 μm and a height of about 100 μm. Thevolume of microwell 5350 is about 3 nL. Printed on the bottom surface ofmicrowell 5350 is a plurality of spatially addressed oligonucleotides5410. Spatially addressed oligonucleotides 5410 include a unique spatialaddress sequence for each microwell 5330.

FIGS. 55A and 55B illustrate an example of a process 5500 of fabricatingmicrowell substrate 5325 of microwell reactor overlay 5300 of FIG. 53.In this example only a portion of microwell substrate 5325 is shown.

In a first step and referring now to FIG. 55A, microwell substrate 5325includes a glass substrate 5510. In one example, glass substrate 5510 isa hydrophilic glass substrate that is about 1 mm thick. A polyimidelayer 5515 is disposed on the surface of glass substrate 5510. In oneexample, polyimide layer 5515 is a black Kapton layer that is about 100μm thick. A hydrophobic layer 5520 is disposed on the surface ofpolyimide layer 5515. In one example, hydrophobic layer 5520 is formedby silanizing polyimide layer 5515.

In a next step and referring now to FIG. 55B, a plurality of microwells5525 are formed in polyimide layer 5515. In this example, two microwells5525 are shown, but any number and arrangement of microwells 5525 may beused. In one example, microwells 5525 are formed in polyimide layer 5515using precise ultraviolet laser ablation to remove portions of polyimidelayer 5515 and hydrophobic layer 5520. In this example, microwells 5525are about 200 μm in diameter. Table 2 below shows other examples ofsuitable dimensions for microwell 5525. Microwells 5525 are hydrophilicregions that function as reaction chambers for performing biochemicalreactions. Because of the presence of hydrophobic layer 5520 in theinterstitial regions between microwells 5525, lateral diffusion ofreaction components or products is eliminated or substantially reduced.The relatively small volume of microwell 5525 provide for small reactionvolume and higher efficiency reactions. Compartmentalization(confinement) of spatially addressed oligonucleotides within microwells5525 also obviates the need for process steps that may be required torelease printed oligonucleotides from the surface of a glass substrate.

TABLE 2 Microwell dimensions Diameter (μm) Height (μm) Volume (nL) 200100 3.1 500 100 19.6 1000 100 78.6

FIG. 56 illustrates a side view of an example of a microwell structure5600 for capture and spatial compartmentalization of nucleic acids froma tissue sample. Microwell structure 5600 includes a substrate 5610. Inone example, substrate 5610 is a glass slide. Substrate 5610 includes anarray of microwells 5615. In one example, microwells 5615 are formed insubstrate 5610 using an etching process, such as by precise ultravioletlaser ablation. Microwells 5615 are hydrophilic regions, while theinterstitial regions between microwells 5615 are hydrophobic regions.Microwells 5615 function as reaction chambers for performing biochemicalreactions (e.g., reverse transcription of RNA to cDNA, amplification ofgenomic DNA). Because the interstitial regions between microwells 5615are hydrophobic, lateral diffusion of reaction components or productsaway from microwells 5615 is eliminated or substantially reduced. In oneexample, microwells 5615 have a diameter of about 1 mm and a height ofabout 100 μm. The spacing between microwells 5615 is about 1 mm.

Deposited in each microwell 5615 is a quantity of gel material 5620. Thequantity of gel material 5620 deposited in each microwell 5615 isselected such that as gel material 5620 is subsequently hydrated, gelmaterial 5620 swells to fill and protrude from microwells 5615 withoutcontacting adjacent microwells 5615. In one example, gel material 5620is a hydrogel material such aspoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZAM) thatis functionalized with covalently linked, spatially addressed captureoligonucleotides (not shown). The spatially addressed captureoligonucleotides (not shown) include a spatial address sequence that isunique for each microwell 5615. The spatially addressed captureoligonucleotides also include a capture sequence for capture of nucleicacids (e.g., RNA or genomic DNA) from a tissue section. The capturesequence can be, for example, a gene-specific capture sequence or auniversal capture sequence. Gel material 5620 can be deposited in eachmicrowell 5615 by printing (e.g., contact printing or piezoelectricprinting).

FIG. 57 illustrates a flow diagram of an example of a method 5700 ofcapturing nucleic acids from a tissue section using microwell structure5600 of FIG. 56 for preparation of a sequencing library. Method 5700includes, but is not limited to, the following steps.

At a step 5710, a quantity of gel material 5620 is deposited in eachmicrowell 5615 of microwell structure 5600, such that each microwell5615 includes gel material 5620 that has a unique spatial addresssequence therein. In some embodiments, microwell structure 5600 that hasgel material 5620 deposited therein can be stored for a period of timeprior to use.

At a step 5715, at time-of-use, microwell structure 5600 is immersed ina biochemical reaction solution to hydrate gel material 5620. In someembodiments, the biochemical reaction solution includes reversetranscriptase and reaction components to synthesize cDNA from targetedRNA in a tissue sample. In some embodiments, the biochemical reactionsolution includes DNA polymerase and reaction components to producemultiple genomic DNA amplicons from targeted genes in a tissue sample.Microwell structure 5600 is immersed in the biochemical reactionsolution for a period of time sufficient for hydration of gel material5620. As gel material 5620 is hydrated, gel material 5620 swells to filland protrude from microwells 5615. Microwell structure 5600 is thenremoved from the biochemical reaction solution. Because the interstitialregion between microwells 5615 is hydrophobic, no biochemical reactionsolution remains between microwells 5615. The spatially addressedcapture oligonucleotides covalently bound to gel material 5620 arelocalized at each microwell 5615.

At a step 5720, a tissue section is placed atop microwell structure 5600such that the tissue section is in contact with the hydrated gelmaterial 5620 that has spatially addressed capture oligonucleotidestherein. In one example, the tissue section is a semi-permeablized FFPEtissue section. As the tissue section contacts hydrated gel material5620 that has spatially addressed capture oligonucleotides thereon, thebiochemical reaction is initiated. Because the interstial regionsbetween microwells 5615 are hydrophobic, reaction components andproducts are localized at each microwell 5615.

At a step 5725, the reaction products (e.g., cDNA or DNA) are removedfrom gel material 5620. The reaction products may be purified from thePAZAM using bead purification methods. At a step 5730, a sequencinglibrary is prepared.

FIG. 58A illustrates a side view of an example of a pin system 5800 fortissue excision and preparation of a spatially addressed nucleic acidlibrary. Pin system 5800 includes a pin structure 5810 and a microwellblock 5815. Pin structure 5810 includes a substrate 5820. In oneexample, substrate 5820 is a glass substrate. Protruding from substrate5820 is an array of pins 5825. In one example, pins 5825 are formed of aglass material. In one example, pins 5825 have a diameter of about 100μm and a height of from about 10 μm to about 20 μm. The spacing betweenpins 5825 is about 100 μm. At the end of each pin 5825 is an excisionsurface 5830. Excision surface 5830 can be any shape that facilitatesremoval of a tissue sample from a tissue section. Examples of differentexcision surfaces are described in more detail with reference to FIG.58B. Pins 5825 can be coated with a substance (not shown) to facilitateadherence of the tissue to pins 5825. In one example, pins 5825 can becoated with poly-lysine. In another example, pins 5825 are coated withan adhesive material. In yet another example, pins 5825 are coated withPAZAM. Substrate 5820 also includes alignment holes 5835. In thisexample, two alignment holes 5835 are shown, but any number of alignmentholes 5835 may be used. Alignment holes 5835 are used to align pinstructure 5810 with microwell block 5815.

Microwell block 5815 includes a substrate 5840. Substrate 5840 includesan array of microwells 5845. Microwells 5845 are arranged to align withpins 5825 in pin structure 5810. Microwells 5845 are loaded with aquantity of a biochemical reaction mixture 5850. In various embodiments,biochemical reaction mixture 5850 includes spatially addressed captureoligonucleotides for targeting and tagging nucleic acids in a tissuesample. The spatially addressed capture oligonucleotides (not shown) inbiochemical reaction mixture 5850 include a spatial address sequencethat is unique for each microwell 5845. In some embodiments, biochemicalreaction mixture 5850 includes spatially addressed captureoligonucleotides, reverse transcriptase and reaction components tosynthesize cDNA from targeted RNA in a tissue sample. In someembodiments, biochemical reaction mixture 5850 includes spatiallyaddressed capture oligonucleotides, DNA polymerase and reactioncomponents to produce multiple genomic DNA amplicons from targeted genesin a tissue sample. In some embodiments, biochemical reaction mixture5850 is a solution of about 1 uL in volume. In some embodiments,biochemical reaction mixture 5850 is a dehydrated reaction mixture orportion thereof that is rehydrated prior to using microwell block 5815.Microwells 5845 with biochemical reaction mixture 5850 therein arecovered with a pierceable film 5855, such as foil. Microwell block 5815also includes alignment pins 5860. In this example, two alignment pins5860 are shown, but any number of alignment pins 5860 may be used.Microwell block 5815 is aligned with pin structure 5810 by fittingalignment pins 5860 of microwell block 5815 into alignment holes 5835 ofpin structure 5810.

FIG. 58B illustrates examples of different excision surfaces 5830 forpins 5825 on pin structure 5810 of FIG. 58A. In one example, excisionsurface 5830 a is concave in shape. In another example, excision surface5830 b is “v” shaped. In yet another example, excision surface 5830 chas a relatively shallow jagged edge shape. In yet another example,excision surface 5830 d has a relatively deep jagged edge shape.

FIG. 59 illustrates a flow diagram of an example of a method 5900 ofcapturing nucleic acids from a tissue section using pin system 5800 ofFIG. 58A for preparation of a sequencing library. In this example, pinstructure 5810 is used as a “touch” pin structure to contact a tissuesection mounted on a solid substrate and remove tissue samples forsubsequent delivery to microwell block 5815 for capture of nucleicacids. Method 5900 includes, but is not limited to, the following steps.

At a step 5910, tissue samples from a tissue section are collected usingpin structure 5810. For example, a tissue section on a glass slide iscontacted with pins 5825 such that samples of tissue adhere to excisionsurfaces 5830. As pin structure 5810 is removed from the surface of thetissue section, adherent tissue samples are removed from the tissuesection. In one example, the tissue section is a semi-permeablized FFPEtissue section.

At a step 5915, pin structure 5810 that has tissue samples thereon ismoved to microwell block 5815. For example, pin structure 5810 isaligned with and mated to microwell block 5815. In so doing, pierceablefilm 5855 is ruptured and pins 5825 that have tissue samples thereon areimmersed in biochemical reaction mixture 5850 within microwells 5845.

At a step 5920, in an incubation period, the biochemical reaction isperformed.

At a step 5925, pin structure 5810 is removed from microwell block 5815and the biochemical reaction mixture that has reaction products (e.g.,cDNA or genomic DNA amplicons) therein is collected from microwells5845.

At a step 5930, a sequencing library is prepared.

FIG. 60 illustrates side views of pin system 5800 of FIG. 58A and showspictorially the step 5910 and 5915 of method 5900 of FIG. 59. Namely, atstep 5910 a tissue section 6010 (e.g., an FFPE tissue section) atop aglass slide 6015 is contacted with pin structure 5815 of pin system5800. Tissue in contact with excision surfaces 5830 adhere to pins 5825.As pin structure 5815 is removed from the surface of tissue section6010, adherent tissue samples 6020 are removed from the tissue section.

At step 5915, pin structure 5810 that has tissue samples 6020 thereon ismoved to microwell block 5815. For example, pin structure 5810 isaligned with and mated to microwell block 5815. In so doing, pierceablefilm 5855 is ruptured and pins 5825 that have tissue samples 6020thereon are immersed in biochemical reaction mixture 5850 withinmicrowells 5845.

FIG. 61 illustrates a flow diagram of another example of a method 6100of capturing nucleic acids from a tissue section using pin system 5800of FIG. 58A for preparation of a sequencing library. In this example,pin structure 5810 is used as a “push” pin structure to contact a tissuesection mounted on a pierceable substrate and push tissue samplesdirectly into microwells 5845 for capture of nucleic acids. Method 6100includes, but is not limited to, the following steps.

At a step 6110, a tissue section mounted on a pierceable substrate isplaced on microwell block 5815; namely, atop pierceable film 5855 ofmicrowell block 5815. In one example, the tissue section is asemi-permeablized FFPE tissue section.

At a step 6115, pin structure 5810 is moved to microwell block 5815. Forexample, pin structure 5810 is aligned with and mated to microwell block5815. In so doing, both the pierceable substrate that has the tissuesection thereon and pierceable film 5855 are ruptured and pins 5825 thathave tissue samples thereon are immersed in biochemical reaction mixture5850 within microwells 5845.

At a step 6120, in an incubation period, the biochemical reaction isperformed.

At a step 6125, pin structure 5810 is removed from microwell block 5815and the biochemical reaction mixture with reaction products (e.g., cDNAor genomic DNA amplicons) therein is collected from microwells 5845.

At a step 6130, a sequencing library is prepared.

FIG. 62 illustrates side views of pin system 5800 of FIG. 58A and showspictorially the steps 6110 and 6115 of method 6100 of FIG. 61. Namely,at step 6110, a tissue section 6210 (e.g., an FFPE tissue section)mounted on a pierceable substrate 6215 is placed atop microwell block5815. At step 6115, pin structure 5810 is moved to microwell block 5815.For example, pin structure 5810 is aligned with and mated to microwellblock 5815. In so doing, both pierceable substrate 6215 that has tissuesection 6210 thereon and pierceable film 5855 are ruptured and pins 5825that have tissue samples 6225 thereon are immersed in biochemicalreaction mixture 5850 within microwells 5845.

FIG. 63 illustrates a prespective view of a capillary “microreactor”system 6300 for capture of nucleic acids from a tissue section forpreparation of a spatially addressed nucleic acid library. Capillarymicroreactor system 6300 includes a plurality of capillary tubes 6310.In one embodiment, the capillary tubes 6310 may used individually tocollect and process a sample or may be bundled in an array and used as aunit. In this example, 4 capillary tubes 6310 are shown, but any numberof capillary tubes 6310 may be used. Capillary tubes 6310 have a samplecontact end 6315 and a non-contact end 6320. Sample contact end 6315 ofeach capillary tube 6310 includes a small protrusion 6325. In oneexample, capillary tubes 6310 are about 100 μm in diameter. Dried on theinner surface of each capillary tube 6310 is a quantity of spatiallyaddressed capture oligonucleotides (not shown) for targeting and taggingnucleic acids from a tissue section. The spatially addressed captureoligonucleotides (not shown) include a spatial address sequence that isunique for each capillary tube 6310. The spatially addressed captureoligonucleotides also include a capture sequence for capture of nucleicacids (e.g., RNA or genomic DNA) from a tissue section. The capturesequence can be, for example, a gene-specific capture sequence or auniversal capture sequence.

At point-of-use, capillary tubes 6310 are filled with a biochemicalreaction solution 6330. In one example, capillary tubes 6310 are filledwith biochemical reaction solution 6330 by “wicking” or capillaryaction. In some embodiments, biochemical reaction solution 6330 includesreverse transcriptase and reaction components to synthesize cDNA fromtargeted RNA in a tissue sample. In some embodiments, biochemicalreaction solution 6330 includes DNA polymerase and reaction componentsto produce genomic DNA amplicons from targeted genes in a tissue sample.

Capillary tubes 6310 are “stamped” onto and press into a tissue section6335 mounted on a substrate 6340. In one example, tissue section 6335 isa semi-permeablized FFPE tissue section. As capillary tubes 6310 arepressed into tissue section 6335, a sample of tissue is pressed intocapillary tube 6310 via protrusion 6325. In some embodiments, an inertsubstrate (not shown) is positioned between tissue section 6335 andsubstrate 6340. The inert substrate is used to seal sample contact ends6315 of capillary tubes 6310.

FIG. 64 illustrates a flow diagram of an example of a method 6400 ofcapturing nucleic acids from a tissue section using capillarymicroreactor system 6300 of FIG. 63 for preparation of a sequencinglibrary. Method 6400 includes, but is not limited to, the followingsteps.

At a step 6410, at point-of-use, biochemical reaction solution 6330 isloaded via capillary action into capillary tubes 6310.

At a step 6415, capillary tubes 6310 that have biochemical reactionsolution 6330 therein are stamped onto and pressed into tissue section6335 mounted on substrate 6340 to collect tissue samples.

At a step 6420, capillary tubes 6310 are removed from substrate 6340 andsealed at sample contact end 6315 to prevent evaporation. In someembodiments, capillary tubes 6310 are removed from substrate 6340 andsample contact ends 6315 are stamped onto an inert substrate to sealagainst evaporation. In some embodiments, an inert substrate ispositioned between tissue section 6335 and substrate 6340. As capillarytubes 6310 are pressed into and through tissue section 6335, samplecontact ends 6315 are pressed into the inert substrate and sealedagainst evaporation.

At a step 6425, non-contact ends 6320 of capillary tubes 6310 are sealedagainst evaporation. In some embodiments, non-contact ends 6320 arestamped onto an inert substrate to seal against evaporation.

At a step 6430, in an incubation period, the biochemical reaction isperformed.

At a step 6435, the biochemical reaction solution with reaction products(e.g., cDNA or genomic DNA amplicons) therein is collected fromcapillary tubes 6310 and pooled.

At a step 6440, a sequencing library is prepared.

4.9 Serial Section DNA/RNA Workflow

A pool of spatially addressed random primer oligonucleotides may be usedin a serial DNA/RNA workflow for spatial detection and analysis of DNAand RNA in a tissue sample. For example, a spatially addressed randomprimer oligonucleotide may include a random primer sequence, a spatialaddress sequence, an SBS primer sequence and a biotin label as describedabove for spatially addressed PCR primer 5100 of FIG. 51. A pool ofspatially addressed random primer oligonucleotides is used in an in situwhole genomic DNA amplification reaction to amplify and spatially indexDNA in one serial section of the tissue sample. The amplified andspatially indexed DNA is then used to generate a sequencing library. Thesame pool of spatially addressed random primer oligonucleotides is usedin an in situ RT reaction to synthesize and spatially index cDNA fromRNA in a second serial section of the tissue sample. The spatiallyindexed cDNA is then used to generate a second sequencing library. Thecombined DNA/RNA workflow provides for increased data output from asingle tissue sample.

4.10 Droplet Actuator Configured for Spatial Detection and Analysis ofNucleic Acids

FIG. 65 illustrates a side view of a portion of droplet actuator 6500that is configured for spatial detection and analysis of nucleic acidsin a tissue sample. Droplet actuator 6500 includes a bottom substrate6510 that is separated from a top substrate 6515 by a droplet operationsgap 6520. Droplet operations are conducted in droplet operations gap6520 on a droplet operations surface. Bottom substrate 6510 includes anarrangement of droplet operations electrodes 6525 (e.g., electrowettingelectrodes). Droplet operations are conducted atop droplet operationselectrodes 6525 on a droplet operations surface. Top substrate 6515includes a reference electrode 6530. Top substrate 6515 also includes arecessed area 6535 that is of sufficient size and shape to accommodate apore sheet 6540. Pore sheet 6540 is described in more detail withreference to FIG. 66. Associated with recessed area 6535 is anelectrophoresis electrode 6545. Bottom substrate 6510 includes acorresponding electrophoresis electrode 6550. Electrophoresis electrodes6545 and 6550 are connected to a voltage source 6555. Pore sheet 6540,electrophoresis electrodes 6545 and 6550 are configured forelectrophoretic transfer and capture of nucleic acids in a tissue samplesuch that spatial orientation is maintained. Further, pore sheet 6540 isinstalled in recessed area 6535 such that there is a certain amount ofspace 6536 between the top of pore sheet 6540 and electrophoresiselectrode 6545.

A hydrophobic layer 6560 is disposed on the surface of top substrate6515 that is facing droplet operations gap 6520. Similarly, anotherhydrophobic layer 6565 is disposed on the surface of bottom substrate5610 that is facing droplet operations gap 6520.

FIG. 66 illustrates a side view of pore sheet 6540 of droplet actuator6500 of FIG. 65. Pore sheet 6540 includes a substrate 6610 that has atop surface 6615 and a bottom surface 6620. Substrate 6610 includes anarray of pores 6625. Pores 6625 are through-holes (e.g., micro-pores) insubstrate 6610 that extent through substrate 6610 from top surface 6615to bottom surface 6620. Each pore 6625 is filled with a gel or asolution or a reaction matrix that includes a plurality of uniquespatially addressed capture probes (not shown). Each spatial addresscorresponds to the position of the capture probes on pore sheet 6540.The position of each pore 6625 may be correlated with a position in atissue sample. Bottom surface 6620 of substrate 6610 may include ahydrophobic layer (not shown) to facilitate transport of a droplet topore sheet 6540 and away from pore sheet 6540.

FIGS. 67A and 67B illustrate side views of droplet actuator 6500 of FIG.65 and show a process 6700 of isolating nucleic acid in a tissue samplefor spatial detection and analysis. Process 6700 is an example of anucleic acid isolation protocol wherein nucleic acids in a tissue sampleare electrophoretically transferred to an array of micro-porescontaining unique spatially addressed capture probes, tagged with theunique spatial address sequence and transferred to a droplet forsubsequent processing on the droplet actuator. Process 6700 includes,but is not limited to, the following steps.

In one step and referring now to FIG. 67A, a tissue sample 6710 mountedon a sample substrate 6715 is positioned in recessed area 6535; namely,in space 6536 atop pore sheet 6540. In one example, tissue sample 6710is an FFPE tissue section. In one example, tissue sample 6710 is placeddirectly on the surface of pore sheet 6540. In another example, tissuesample 6710 is placed on a substance (not shown) such as a gel or a thinbuffer layer separating tissue sample 6710 from pore sheet 6540 tofacilitate electrophoretic transfer of nucleic acids from tissue sample6710 into pore sheet 6540. A droplet 6720 is transported using dropletoperations to a certain droplet operations electrode 6525 aligned withpore sheet 6540. A voltage is applied from voltage source 6555 toelectrophoresis electrodes 6545 and 6550, creating an electric field. Inthe presence of the electric field, a plurality of nucleic acids 6725are transferred from tissue sample 6710 into pores 6625 of pore sheet6540. Nucleic acid 6725 in each pore 6625 of pore sheet 6540 is taggedwith a spatial address sequence (not shown) that corresponds to theposition of each pore 6625 on pore sheet 6540.

In another step and referring now to FIG. 67B, after a period of timesufficient for capture of nucleic acids 6725 onto spatially addressedcapture probes contained in each pore 6625, nucleic acids 6725 areelectrophoretically transferred into droplet 6720. Droplet 6720 withtagged nucleic acids 6725 therein is transported using dropletoperations away from pore sheet 6540 for subsequent processing steps ondroplet actuator 6500. For example, droplet actuator 6500 may include areaction zone (not shown) for performing processing steps in asequencing library preparation protocol (e.g., reverse transcription ofRNA to cDNA, exonuclease I digestion, PCR amplification, and Nexteralibrary preparation). Droplet actuator 6500 may also include asequencing zone (not shown) for performing a DNA sequencing protocol(e.g., an SBS protocol). The sequencing zone may be, for example, asequencing flow cell.

4.11 Spatial Tissue Profiling Based on DNA Tagmentation

“Tagmentation,” as used herein, is a process of transposase mediatedfragmentation and tagging. Tagmentation often involves the modificationof DNA by a transposome complex comprising transposase enzyme complexedwith adaptors comprising a transposon end sequence. Tagmentation resultsin the simultaneous fragmentation of DNA and ligation of the adapters tothe 5′ ends of both strands of DNA duplex fragments.

This disclosure is based, in part, on the realization that tagmentationcan be efficiently used to spatially address nucleic acids from a tissuesample on a capture array.

FIG. 68 illustrates a flow diagram of an example of a method 6800 ofprofiling genomic DNA in a tissue sample using tagmentation of the DNA.In this example, method 6800 is used for profiling SNVs of interest ingenomic DNA. Method 6800 can comprise, but is not limited to, some orall of the following steps.

At a step 6810, a glass substrate is printed with spatially addressedoligonucleotides to form an array of spatial features. In someembodiments, the spatially addressed oligonucleotides can be printed ona 2 cm×2 cm coverslip to form an array of spatial features that are 100μm in diameter on a pitch of 35 μm. In some embodiments, the spatiallyaddressed oligonucleotides can be printed in microwells fabricated onthe surface of a glass slide. The spatially addressed oligonucleotidescan be printed on a coverslip or glass slide using, for example,commercially available printing technologies. The spatially addressedoligonucleotides can comprise a linker sequence, an SBS primer sequence,a spatial address sequence, and a 19 bp Mosaic End (ME) sequence asdescribed in more detail with reference to FIG. 69A. In someembodiments, the ME sequence is a Tn5 transposase recognition sequence.In some embodiments, the ME sequence is a Mu transposase recognitionsequence. The spatially addressed oligonucleotides can further comprisea modification at the 5′ end of the molecule for reversible attachmentto the coverslip. In some embodiments, the spatially addressedoligonucleotides can comprise a 5′ disulfide modification as describedfor spatially addressed capture probe 3100 of process 3200 of FIGS. 32A,32B, and 32C. In some embodiments, the spatially addressed PCR primerscan include a 5′ photocleavable linker as described for spatiallyaddressed capture probe 3100 of process 3300 of FIGS. 33A, 33B, and 33C.

At a step 6815, a reverse complement oligonucleotide sequence ishybridized to the ME sequence to form a region of double stranded DNA.

At a step 6820, a transposase enzyme solution is added onto the surfaceof the spatially addressed oligonucleotide array to form a transposomehomodimer at each region of double stranded DNA. In some embodiments,the transposase enzyme solution comprises Tn5. In some embodiments, thetransposase enzyme solution comprises Mu.

At a step 6825, a tissue section is placed on the array, such that thesurface of the array substrate with the spatially addressedoligonucleotides and transposome homodimers thereon is in contact withthe tissue section. In one example, the tissue section is an FFPE tissuesection.

At a step 6830, the double stranded DNA is tagmented with a transposomecomplex. Methods, compositions, and kits for treating nucleic acid, andin particular, methods and compositions for fragmenting and tagging DNAusing transposon compositions are described in detail, for example, inUS2010/0120098 and US2011/0287435, which are hereby incorporated byreference in their entireties.

At a step 6835, the tagmented DNA is amplified using a gene specificprimer and a universal primer that includes a complementary region tothe SBS primer sequence to a generate tagmented genomic DNA library.

At a step 6840, the tagmented genomic DNA library is sequenced.

FIGS. 69A, 69B, and 69C illustrate the steps of the method 6800 of FIG.68. At step 6810 (see FIG. 69A), an array surface 6910 is printed withspatially addressed oligonucleotides 6915 for spatial indexing andtagmentation of whole genomic DNA. In this example, a single spatiallyaddressed oligonucleotide 6915 is shown, but any number of 6915oligonucleotides can be immobilized on the array surface 6010, Spatiallyaddressed oligonucleotide 6015 includes a linker region 6920, an SBSprimer region 6925, a spatial address region 6930, and a ME region 6935.Spatial address region 6930 comprises a unique sequence for each capturesite (spatial feature) on an array. The 19 bp ME sequence of ME region6935 or the transposon end is described in detail, e.g., inUS2010/0120098 and US2011/0287435. Methods, compositions, and kits fortreating nucleic acid, and in particular, methods and compositions forfragmenting and tagging DNA using transposon compositions are describedin detail, e.g., in US2010/0120098 and US2011/0287435.

The linker region 6920 in this example comprises a cleavable sequencethat can be used to release captured nucleic acid from array surface6910 such that spatial address region 6930 is included in the releasednucleic acid and the nucleic acid is “tagged.” SBS primer region 6925comprise an SBS primer sequence (e.g., SBS12 or SBS3) that can be usedin a sequencing-by-synthesis (SBS) process. SBS primer region 6925 canalso be used in an amplification reaction to generate a sequencinglibrary as described in more detail with reference to FIG. 12 and FIG.13.

At step 6815 (see FIG. 69A), an ME reverse complement sequence 6940 ishybridized to the ME region 6935.

At step 6820 (see FIG. 69B), a transposase enzyme solution (not shown)is added onto the array surface 6910 to form a transposome homodimer6945 at each region of double stranded DNA. In some embodiments, thetransposome ends comprise Mu transposome ends and the transposase is Mutransposase. In some embodiments, the transposome ends comprise Tn5transposome ends and the transposase is Tn5 transposase.

At step 6825 (see FIG. 69B), a tissue section 6950 is placed atop thearray surface 6910 such that the spatially addressed oligonucleotides6915 and transposome homodimers 6945 thereon is in contact with thetissue section 6950. In some embodiments, tissue section 6950 is an FFPEtissue section. Tissue section 6950 includes a cell 6955. Cell 6955includes a genomic DNA molecule 6960. Genomic DNA molecule 6960 mayinclude a single nucleotide variation (SNV) 6965.

At step 6830 (see FIG. 69C), genomic DNA 6960 is tagmented. For example,genomic DNA 6960 is tagmented such that SNV 6965 is “A” upstream of atagmentation event or “B” downstream of a tagmentation event.

At step 6835 (see FIG. 69C), tagmented genomic DNA 6960 is amplified.For example, if SNV 6965 is “A” upstream of a tagmentation event,genomic DNA 6960 is amplified using a gene-specific primer 6970 and auniversal primer 6975 that includes a complementary region to the SBSprimer region 6925. If SNV 6965 is “B” downstream of a tagmentationevent, genomic DNA 6960 is amplified using a gene-specific primer 6980and the universal primer 6975.

At step 6840 (not shown in FIGS. 69A, 69B, and 69C), the PCR product issequenced.

In another aspect, provided herein is a capture array for spatialdetection and analysis of nucleic acids in a tissue sample, comprising acapture site comprising a capture probe (e.g., 6915) comprising aspatial address region (e.g., 6930), and a transposon end (TE) region(e.g., 6935). In some embodiments, the capture probe further comprises acleavable region (e.g., 6920) and an SBS primer binding region (e.g.,6925). In some embodiments, the transposon end region is hybridized to areverse-complementary oligonucleotide (e.g., 6940) to form adouble-stranded transposon end region. In some embodiments, the TEregion comprises an ME sequence.

In some embodiments, the capture array further comprises a transposaseto form a transposome (e.g., 6945).

In some embodiments the transposome ends comprise Mu transposome endsand the transposase is Mu transposase. In some embodiments thetransposome ends comprise Tn5 transposome ends and the transposase isTn5 transposase.

In another aspect, provided herein is a method for spatial detection andanalysis of nucleic acids in a tissue sample, comprising providing acapture array described herein. In some embodiments, the capture arraycomprises a capture site comprising a capture probe (e.g., 6915)comprising a spatial address region (e.g., 6930), and a transposon end(TE) region (e.g., 6935). In some embodiments, the capture probe furthercomprises a cleavable region (e.g., 6920) and an SBS primer bindingregion (e.g., 6925).

In some embodiments, the method further comprises contacting the capturearray with an oligonucleotide that is a reverse-complement of the TEregion (e.g., 6940) to form a double-stranded transposon end region.

In some embodiments, the method further comprises contacting the capturearray with a transposase to form a transposome (e.g., 6945). In someembodiments the transposome ends comprise Mu transposome ends and thetransposase is Mu transposase. In some embodiments the transposome endscomprise Tn5 transposome ends and the transposase is Tn5 transposase.

In some embodiments, the method further comprises contacting the capturearray with a tissue sample such that the position of a capture site onthe array can be correlated with a position in the tissue sample; andallowing a tagmentation reaction to occur between the genomic DNA of thetissue sample and the transposome at the capture site. In someembodiments, the genomic DNA comprises a SNV.

In some embodiments, the method further comprises analyzing the sequenceof the tagmented DNA. In some embodiments, sequencing the tagmented DNAcomprises performing a sequencing reaction using a combination of agene-specific primer and a universal primer. IN some embodiments,analyzing the sequence of the tagmented DNA comprises detecting the SNV.

In some embodiments, the method further comprises correlating thesequence of the tagmented DNA to the position of the genomic DNA in thetissue sample. In some embodiments, correlating the sequence of thetagmented DNA comprises correlating the SNV with a position in thetissue sample.

4.12 Sequencing Methods

The methods described herein can be used in conjunction with a varietyof nucleic acid sequencing techniques. Particularly applicabletechniques are those wherein nucleic acids are attached at fixedlocations in an array such that their relative positions do not changeand wherein the array is repeatedly imaged. Embodiments in which imagesare obtained in different color channels, for example, coinciding withdifferent labels used to distinguish one nucleotide base type fromanother are particularly applicable. In some embodiments, the process todetermine the nucleotide sequence of a target nucleic acid can be anautomated process. Preferred embodiments include sequencing-by-synthesis(“SBS”) techniques.

“Sequencing-by-synthesis (“SBS”) techniques” generally involve theenzymatic extension of a nascent nucleic acid strand through theiterative addition of nucleotides against a template strand. Intraditional methods of SBS, a single nucleotide monomer can be providedto a target nucleotide in the presence of a polymerase in each delivery.However, in the methods described herein, more than one type ofnucleotide monomer can be provided to a target nucleic acid in thepresence of a polymerase in a delivery.

SBS can utilize nucleotide monomers that have a terminator moiety orthose that lack any terminator moieties. Methods utilizing nucleotidemonomers lacking terminators include, for example, pyrosequencing andsequencing using γ-phosphate-labeled nucleotides, as set forth infurther detail below. In methods using nucleotide monomers lackingterminators, the number of nucleotides added in each cycle is generallyvariable and dependent upon the template sequence and the mode ofnucleotide delivery. For SBS techniques that utilize nucleotide monomershaving a terminator moiety, the terminator can be effectivelyirreversible under the sequencing conditions used as is the case fortraditional Sanger sequencing which utilizes dideoxynucleotides, or theterminator can be reversible as is the case for sequencing methodsdeveloped by Solexa (now Illumina, Inc.).

SBS techniques can utilize nucleotide monomers that have a label moietyor those that lack a label moiety. Accordingly, incorporation events canbe detected based on a characteristic of the label, such as fluorescenceof the label; a characteristic of the nucleotide monomer such asmolecular weight or charge; a byproduct of incorporation of thenucleotide, such as release of pyrophosphate; or the like. Inembodiments, where two or more different nucleotides are present in asequencing reagent, the different nucleotides can be distinguishablefrom each other, or alternatively, the two or more different labels canbe the indistinguishable under the detection techniques being used. Forexample, the different nucleotides present in a sequencing reagent canhave different labels and they can be distinguished using appropriateoptics as exemplified by the sequencing methods developed by Solexa (nowIllumina, Inc.).

Preferred embodiments include pyrosequencing techniques. Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into the nascent strand (Ronaghi, M.,Karamohamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996)“Real-time DNA sequencing using detection of pyrophosphate release.”Analytical Biochemistry 242(1), 84-9; Ronaghi, M. (2001) “Pyrosequencingsheds light on DNA sequencing.” Genome Res. 11(1), 3-11; Ronaghi, M.,Uhlen, M. and Nyren, P. (1998) “A sequencing method based on real-timepyrophosphate.” Science 281(5375), 363; U.S. Pat. Nos. 6,210,891;6,258,568 and 6,274,320, the disclosures of which are incorporatedherein by reference in their entireties). In pyrosequencing, releasedPPi can be detected by being immediately converted to adenosinetriphosphate (ATP) by ATP sulfurylase, and the level of ATP generated isdetected via luciferase-produced photons. The nucleic acids to besequenced can be attached to features in an array and the array can beimaged to capture the chemiluminscent signals that are produced due toincorporation of a nucleotides at the features of the array. An imagecan be obtained after the array is treated with a particular nucleotidetype (e.g., A, T, C or G). Images obtained after addition of eachnucleotide type will differ with regard to which features in the arrayare detected. These differences in the image reflect the differentsequence content of the features on the array. However, the relativelocations of each feature will remain unchanged in the images. Theimages can be stored, processed and analyzed using the methods set forthherein. For example, images obtained after treatment of the array witheach different nucleotide type can be handled in the same way asexemplified herein for images obtained from different detection channelsfor reversible terminator-based sequencing methods.

In another exemplary type of SBS, cycle sequencing is accomplished bystepwise addition of reversible terminator nucleotides containing, forexample, a cleavable or photobleachable dye label as described, forexample, in International Patent Pub. No. WO 04/018497 and U.S. Pat. No.7,057,026, the disclosures of which are incorporated herein byreference. This approach is being commercialized by Solexa (now IlluminaInc.), and is also described in International Patent Pub. No. WO91/06678 and International Patent Pub. No. WO 07/123,744, each of whichis incorporated herein by reference. The availability offluorescently-labeled terminators in which both the termination can bereversed and the fluorescent label cleaved facilitates efficient cyclicreversible termination (CRT) sequencing. Polymerases can also beco-engineered to efficiently incorporate and extend from these modifiednucleotides.

Preferably in reversible terminator-based sequencing embodiments, thelabels do not substantially inhibit extension under SBS reactionconditions. However, the detection labels can be removable, for example,by cleavage or degradation. Images can be captured followingincorporation of labels into arrayed nucleic acid features. Inparticular embodiments, each cycle involves simultaneous delivery offour different nucleotide types to the array and each nucleotide typehas a spectrally distinct label. Four images can then be obtained, eachusing a detection channel that is selective for one of the fourdifferent labels. Alternatively, different nucleotide types can be addedsequentially and an image of the array can be obtained between eachaddition step. In such embodiments each image will show nucleic acidfeatures that have incorporated nucleotides of a particular type.Different features will be present or absent in the different images duethe different sequence content of each feature. However, the relativeposition of the features will remain unchanged in the images. Imagesobtained from such reversible terminator-SBS methods can be stored,processed and analyzed as set forth herein. Following the image capturestep, labels can be removed and reversible terminator moieties can beremoved for subsequent cycles of nucleotide addition and detection.Removal of the labels after they have been detected in a particularcycle and prior to a subsequent cycle can provide the advantage ofreducing background signal and crosstalk between cycles. Examples ofuseful labels and removal methods are set forth below.

In particular embodiments some or all of the nucleotide monomers caninclude reversible terminators. In such embodiments, reversibleterminators/cleavable fluors can include fluor linked to the ribosemoiety via a 3′ ester linkage (Metzker, Genome Res. 15:1767-1776 (2005),which is incorporated herein by reference). Other approaches haveseparated the terminator chemistry from the cleavage of the fluorescencelabel (Ruparel et al., Proc Natl Acad Sci USA 102: 5932-7 (2005), whichis incorporated herein by reference in its entirety). Ruparel et aldescribed the development of reversible terminators that used a small 3′allyl group to block extension, but could easily be deblocked by a shorttreatment with a palladium catalyst. The fluorophore was attached to thebase via a photocleavable linker that could easily be cleaved by a 30second exposure to long wavelength UV light. Thus, either disulfidereduction or photocleavage can be used as a cleavable linker. Anotherapproach to reversible termination is the use of natural terminationthat ensues after placement of a bulky dye on a dNTP. The presence of acharged bulky dye on the dNTP can act as an effective terminator throughsteric and/or electrostatic hindrance. The presence of one incorporationevent prevents further incorporations unless the dye is removed.Cleavage of the dye removes the fluor and effectively reverses thetermination. Examples of modified nucleotides are also described in U.S.Pat. Nos. 7,427,673, and 7,057,026, the disclosures of which areincorporated herein by reference in their entireties.

Additional exemplary SBS systems and methods which can be utilized withthe methods and systems described herein are described in U.S. PatentPub. No. 2007/0166705, U.S. Patent Pub. No. 2006/0188901, U.S. Pat. No.7,057,026, U.S. Patent Pub. No. 2006/0240439, U.S. U.S. Patent Pub. No.2006/0281109, International Patent Pub. No. WO 05/065814, U.S. PatentPub. No. 2005/0100900, International Patent Pub. No. WO 06/064199,International Patent Pub. No. WO 07/010,251, U.S. U.S. Patent Pub. No.2012/0270305 and U.S. Patent Pub. No. 2013/0260372, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize detection of four different nucleotidesusing fewer than four different labels. For example, SBS can beperformed utilizing methods and systems described in the incorporatedmaterials of U.S. Patent Pub. No. 2013/0079232. As a first example, apair of nucleotide types can be detected at the same wavelength, butdistinguished based on a difference in intensity for one member of thepair compared to the other, or based on a change to one member of thepair (e.g., via chemical modification, photochemical modification orphysical modification) that causes apparent signal to appear ordisappear compared to the signal detected for the other member of thepair. As a second example, three of four different nucleotide types canbe detected under particular conditions while a fourth nucleotide typelacks a label that is detectable under those conditions, or is minimallydetected under those conditions (e.g., minimal detection due tobackground fluorescence, etc.). Incorporation of the first threenucleotide types into a nucleic acid can be determined based on presenceof their respective signals and incorporation of the fourth nucleotidetype into the nucleic acid can be determined based on absence or minimaldetection of any signal. As a third example, one nucleotide type caninclude label(s) that are detected in two different channels, whereasother nucleotide types are detected in no more than one of the channels.The aforementioned three exemplary configurations are not consideredmutually exclusive and can be used in various combinations. An exemplaryembodiment that combines all three examples, is a fluorescent-based SBSmethod that uses a first nucleotide type that is detected in a firstchannel (e.g., dATP having a label that is detected in the first channelwhen excited by a first excitation wavelength), a second nucleotide typethat is detected in a second channel (e.g., dCTP having a label that isdetected in the second channel when excited by a second excitationwavelength), a third nucleotide type that is detected in both the firstand the second channel (e.g., dTTP having at least one label that isdetected in both channels when excited by the first and/or secondexcitation wavelength) and a fourth nucleotide type that lacks a labelthat is not, or minimally, detected in either channel (e.g., dGTP havingno label).

Further, as described in the incorporated materials of U.S. Patent Pub.No. 2013/0079232, sequencing data can be obtained using a singlechannel. In such so-called one-dye sequencing approaches, the firstnucleotide type is labeled but the label is removed after the firstimage is generated, and the second nucleotide type is labeled only aftera first image is generated. The third nucleotide type retains its labelin both the first and second images, and the fourth nucleotide typeremains unlabeled in both images.

Some embodiments can utilize sequencing by ligation techniques. Suchtechniques utilize DNA ligase to incorporate oligonucleotides andidentify the incorporation of such oligonucleotides. Theoligonucleotides typically have different labels that are correlatedwith the identity of a particular nucleotide in a sequence to which theoligonucleotides hybridize. As with other SBS methods, images can beobtained following treatment of an array of nucleic acid features withthe labeled sequencing reagents. Each image will show nucleic acidfeatures that have incorporated labels of a particular type. Differentfeatures will be present or absent in the different images due thedifferent sequence content of each feature, but the relative position ofthe features will remain unchanged in the images. Images obtained fromligation-based sequencing methods can be stored, processed and analyzedas set forth herein. Exemplary SBS systems and methods which can beutilized with the methods and systems described herein are described inU.S. Pat. Nos. 6,969,488, 6,172,218, and 6,306,597, the disclosures ofwhich are incorporated herein by reference in their entireties.

Some embodiments can utilize nanopore sequencing (Deamer, D. W. &Akeson, M. “Nanopores and nucleic acids: prospects for ultrarapidsequencing.” Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D.Branton, “Characterization of nucleic acids by nanopore analysis”. Acc.Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin,and J. A. Golovchenko, “DNA molecules and configurations in asolid-state nanopore microscope” Nat. Mater. 2:611-615 (2003), thedisclosures of which are incorporated herein by reference in theirentireties). In such embodiments, the target nucleic acid passes througha nanopore. The nanopore can be a synthetic pore or biological membraneprotein, such as α-hemolysin. As the target nucleic acid passes throughthe nanopore, each base-pair can be identified by measuring fluctuationsin the electrical conductance of the pore. (U.S. Pat. No. 7,001,792;Soni, G. V. & Meller, “A. Progress toward ultrafast DNA sequencing usingsolid-state nanopores.” Clin. Chem. 53, 1996-2001 (2007); Healy, K.“Nanopore-based single-molecule DNA analysis.” Nanomed. 2, 459-481(2007); Cockroft, S. L., Chu, J., Amorin, M. & Ghadiri, M. R. “Asingle-molecule nanopore device detects DNA polymerase activity withsingle-nucleotide resolution.” J. Am. Chem. Soc. 130, 818-820 (2008),the disclosures of which are incorporated herein by reference in theirentireties). Data obtained from nanopore sequencing can be stored,processed and analyzed as set forth herein. In particular, the data canbe treated as an image in accordance with the exemplary treatment ofoptical images and other images that is set forth herein.

Some embodiments can utilize methods involving the real-time monitoringof DNA polymerase activity. Nucleotide incorporations can be detectedthrough fluorescence resonance energy transfer (FRET) interactionsbetween a fluorophore-bearing polymerase and γ-phosphate-labelednucleotides as described, for example, in U.S. Pat. Nos. 7,329,492 and7,211,414 (each of which is incorporated herein by reference) ornucleotide incorporations can be detected with zero-mode waveguides asdescribed, for example, in U.S. Pat. No. 7,315,019 (which isincorporated herein by reference) and using fluorescent nucleotideanalogs and engineered polymerases as described, for example, in U.S.Pat. No. 7,405,281 and U.S. Patent Pub. No. 2008/0108082 (each of whichis incorporated herein by reference). The illumination can be restrictedto a zeptoliter-scale volume around a surface-tethered polymerase suchthat incorporation of fluorescently labeled nucleotides can be observedwith low background (Levene, M. J. et al. “Zero-mode waveguides forsingle-molecule analysis at high concentrations.” Science 299, 682-686(2003); Lundquist, P. M. et al. “Parallel confocal detection of singlemolecules in real time.” Opt. Lett. 33, 1026-1028 (2008); Korlach, J. etal. “Selective aluminum passivation for targeted immobilization ofsingle DNA polymerase molecules in zero-mode waveguide nano structures.”Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures ofwhich are incorporated herein by reference in their entireties). Imagesobtained from such methods can be stored, processed and analyzed as setforth herein.

Some SBS embodiments include detection of a proton released uponincorporation of a nucleotide into an extension product. For example,sequencing based on detection of released protons can use an electricaldetector and associated techniques that are commercially available fromIon Torrent (Guilford, Conn., a Life Technologies subsidiary) orsequencing methods and systems described in U.S. Patent Pub. No.2009/0026082; U.S. Patent Pub. No. 2009/0127589; U.S. Patent Pub. No.2010/0137143; or U.S. Patent Pub. No. 2010/0282617, each of which isincorporated herein by reference. Methods set forth herein foramplifying target nucleic acids using kinetic exclusion can be readilyapplied to substrates used for detecting protons. More specifically,methods set forth herein can be used to produce clonal populations ofamplicons that are used to detect protons.

The above SBS methods can be advantageously carried out in multiplexformats such that multiple different target nucleic acids aremanipulated simultaneously. In particular embodiments, different targetnucleic acids can be treated in a common reaction vessel or on a surfaceof a particular substrate. This allows convenient delivery of sequencingreagents, removal of unreacted reagents and detection of incorporationevents in a multiplex manner. In embodiments using surface-bound targetnucleic acids, the target nucleic acids can be in an array format. In anarray format, the target nucleic acids can be typically bound to asurface in a spatially distinguishable manner. The target nucleic acidscan be bound by direct covalent attachment, attachment to a bead orother particle or binding to a polymerase or other molecule that isattached to the surface. The array can include a single copy of a targetnucleic acid at each site (also referred to as a feature) or multiplecopies having the same sequence can be present at each site or feature.Multiple copies can be produced by amplification methods such as, bridgeamplification or emulsion PCR as described in further detail below.

The methods set forth herein can use arrays having features at any of avariety of densities including, for example, at least about 10features/cm², 100 features/cm², 500 features/cm², 1,000 features/cm²,5,000 features/cm², 10,000 features/cm², 50,000 features/cm², 100,000features/cm², 1,000,000 features/cm², 5,000,000 features/cm², or higher.

An advantage of the methods set forth herein is that they provide forrapid and efficient detection of a plurality of target nucleic acid inparallel. Accordingly the present disclosure provides integrated systemscapable of preparing and detecting nucleic acids using techniques knownin the art such as those exemplified above. Thus, an integrated systemof the present disclosure can include fluidic components capable ofdelivering amplification reagents and/or sequencing reagents to one ormore immobilized DNA fragments, the system comprising components such aspumps, valves, reservoirs, fluidic lines and the like. A flow cell canbe configured and/or used in an integrated system for detection oftarget nucleic acids. Exemplary flow cells are described, for example,in U.S. Patent Pub. No. 2010/0111768 A1 and U.S. patent application Ser.No. 13/273,666, each of which is incorporated herein by reference. Asexemplified for flow cells, one or more of the fluidic components of anintegrated system can be used for an amplification method and for adetection method. Taking a nucleic acid sequencing embodiment as anexample, one or more of the fluidic components of an integrated systemcan be used for an amplification method set forth herein and for thedelivery of sequencing reagents in a sequencing method such as thoseexemplified above. Alternatively, an integrated system can includeseparate fluidic systems to carry out amplification methods and to carryout detection methods. Examples of integrated sequencing systems thatare capable of creating amplified nucleic acids and also determining thesequence of the nucleic acids include, without limitation, the MiSeq™platform (Illumina, Inc., San Diego, Calif.) and devices described inU.S. patent application Ser. No. 13/273,666, which is incorporatedherein by reference. For example, the MiSeq™ platform may be implementedwith capture probes 5′ CAACGATCGTCGAAATTCGC[target primer] 3′ and 5′[target primer]AGATCGGAAGAGCGTCGTGTA3′ where [target primer] is asequence which is complimentary to a target nucleic acid.

4.13 Concluding Remarks

The foregoing detailed description of embodiments refers to theaccompanying drawings, which illustrate specific embodiments of thepresent disclosure. Other embodiments having different structures andoperations do not depart from the scope of the present disclosure. Thisspecification is divided into sections for the convenience of the readeronly. Headings should not be construed as limiting of the scope of thedisclosure provided herein. The definitions are intended as a part ofthe disclosure provided herein. It will be understood that variousdetails of the present disclosure can be changed without departing fromthe scope of the disclosed embodiments. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

5. EXAMPLES

5.1 Sensitivity of Spatial NGS for Detection of Single NucleotideVariations

This example demonstrates that sensitivity of SNV detection can besubstantially increased using spatially addressed sequencing compared tobulk sequencing.

The sensitivity of SNV detection using bulk sequencing or spatiallyaddressed sequencing data was evaluated using simulated data sets. Thecalculations used to derive a bulk sequencing data set was based on thefollowing assumptions: (1) a typical FFPE section is 1.5 cm×1.5 cm,which is about 225,000,000 μm²; (2) a typical cell is 20×20 μm, whichhas an area of about 400 μm²; (3) the number of cells in a typical FFPEsection is about 563,000 (i.e., 225,000,000 μm² FFPE block÷400 μm²cell=˜563K cells per section); and (4) the packing density of cells inan FFPE section is about 70%, then the number of cells in a typical FFPEsection is about 400,000. Table 3 below shows simulated data for thesensitivity of SNV detection in bulk sequencing. To identify rarepopulations of clonally mutated cells using bulk sequencing, the variantfrequency (% SNV) needs to be above the sequencing error rate, which isabout 1%. For example, 1 variant cell (“Localized mutated cells”) in aFFPE section has a variant frequency (% SNV) of 0.00025 (i.e., (1cell÷400,000 cells in FFPE section)×100)=0.00025% SNV), which is wellbelow the sequencing error rate and is therefore not detectable in bulksequencing data. At least about 4000 cells are required to detect an SNVin bulk sequencing data, e.g., 4096 cells÷400,000 cells in FFPEsection)×100)=1.024, which is above the sequencing error rate.

TABLE 3 Sensitivity of SNV detection in bulk sequencing SNV detectionLocalized sensitivity above mutated cells % SNV (bulk sequence data)sequencing error*? 1 0.00025 No 4 0.001 No 16 0.004 No 64 0.016 No 2560.064 No 1024 0.256 No 2048 0.512 No 4096 1.024 Yes 8192 2.048 Yes *1%sequencing error rate

It was found that sensitivity of SNV detection can be substantiallyincreased using spatially addressed sequencing as exemplified using asimulated data set based on a 1.5 cm×1.5 cm tissue section overlaid on a2 cm×2 cm array. FIG. 70 illustrates a plan view of a spatial addressoverlay 7000. Spatial address overlay 7000 comprises a substrate 7005.In one example, substrate 7005 is a planar glass substrate such as acoverslip that is 2 cm×2 cm in size. Printed on the surface of substrate7005 are a plurality of distinct features (not shown) which are regionscontaining spatially addressed oligonucleotides. An example of a singlespatial feature is described in more detail with reference to FIG. 71.Overlaid on substrate 7005 is a tissue section 7010. In one example,tissue section 7005 is an FFPE tissue section that is 1.5 cm×1.5 cm insize.

FIG. 71 illustrates a plan view of a single spatial feature 7100 onsubstrate 7005 of FIG. 70. In this example, spatial feature 7100 is a100 μm×100 μm square (area=10,000 μm²). Spatial feature 7100 is ofsufficient size to encompass a plurality of cells 7105 contained in atissue sample. Cells 7105 can be normal cells (e.g., 7105 a) or variant(mutated) cells (e.g., 7105 b). In one example, cells 7105 are 20 μm×20μm in size (area=400 μm²) and the cell packing density in the tissuesection is about 70%. Based on these parameters, the number of cells7105 that are encompassed in spatial feature 7100 is about 18 (i.e.,(10,000 μm²÷400*0.7)=˜18).

Table 4 below shows the simulated data for the sensitivity of SNVdetection in spatially addressed sequencing data based on spatialaddress overlay 7000 of FIG. 70 and FIG. 71. For example, 1 variant cell(“Localized mutated cells) in a single spatial feature 3000 has avariant frequency (% SNV) of about 6 (i.e., (1 cell÷18 cells per spatialfeature)*100=6% SNV), which is above the 1% sequencing error rate and istherefore detectable in spatially addressed sequencing data. Incomparison, about 4000 cells or more are required for SNV detection inbulk sequencing data.

TABLE 4 Comparison of sensitivity of SNV detection between spatiallyindexed and bulk sequencing data Bulk sequencing Sequence data tagged byspatial index SNV SNV detection detection sensitivity sensitivityLocalized above % SNV above mutated % SNV (spatial sequencing (spatialsequencing cells† indexing) error*? indexing) error? 1  6 Yes 0.00025 No2 11 Yes 0.0005 No 4 22 Yes 0.001 No 16 89 Yes 0.004 No 32 ~2 spatialindex Yes 0.008 No regions 64 ~ 4 spatial index Yes 0.016 No regions 128~7 spatial index Yes 0.032 No regions 256 ~14 spatial Yes 0.064 No indexregions . . . . . . . . . . . . . . . 4000 ~220 spatial Yes 1 Yes indexregions †Cells per spatial feature; *1% sequencing error rateThe number (x) of spatial features 7100 on substrate 7005 required toachieve a desired level of detection sensitivity can be calculated asfollows: (X+Y array length)=(spatial feature edge×x)+((x−1)×spatialfeature spacing)), where the array length is 20,000 μm, the spatialfeature edge is 100 μm, and the spatial feature spacing is, for example,50 μm; then x=133 spatial features for X dimension and 133 spatialfeatures for Y dimension. The total number of features on substrate 7005(2 cm×2 cm) is 133×133=˜17,689.

What is claimed is:
 1. A method, comprising: (a) providing a capturearray comprising a plurality of capture sites, wherein each capture siteof the plurality of capture sites comprises: a pair of probes that areseparately immobilized on a surface at each capture site, wherein afirst probe of the pair of probes comprises a first primer bindingregion sequence and a spatial address region sequence and does notcomprise a poly-T capture sequence, wherein the spatial address regionsequence of the first probe comprises a sequence of nucleic acids uniqueto each capture site such that the spatial address region sequence ofeach capture site of the plurality of capture sites is differentrelative to one another, wherein a second probe of the pair of probescomprises a second primer binding region sequence and the poly-T capturesequence and does not comprise the spatial address region sequence andwherein the second probe is a same sequence at each of the plurality ofcapture sites, wherein the poly-T capture sequence is configured tohybridize to a target nucleic acid comprising a poly-A tail; (b)contacting the plurality of capture sites of the capture array with atissue sample such that a position of each capture site on the capturearray can be correlated with a position in the tissue sample; (c)subsequent to the contacting, allowing target nucleic acids of thetissue sample, the target nucleic acids comprising the poly-A tail, tohybridize to respective poly-T capture sequences of the second probe ofthe plurality of capture sites to form hybridized second probes; (d)extending the poly-T capture region sequence of the hybridized secondprobes to form first complementary strands of the sample nucleic acids;and (e) linking ends of the first complementary strands to the firstprobes at each capture site to form immobilized first complementarystrands and such that the immobilized first complementary strandcomprises the unique spatial address region sequence of the firstcapture probe associated with each respective capture site and such thatimmobilized first complementary strands at different capture sitescomprise different spatial address region sequences.
 2. The method ofclaim 1, further comprising wherein (e) comprises ligating theimmobilized first complementary strands to the spatial address regionsequence of the first probes at each capture site to immobilize thefirst complementary strands at both ends; (f) releasing the firstcomplementary strands from the surface of the capture array andamplifying the first complementary strands to generate secondcomplementary strands; (g) analyzing a sequence of the secondcomplementary strands; and (h) correlating the sequence of the secondcomplementary strands to the position of the target nucleic acid in thetissue sample.
 3. The method of claim 1, wherein allowing nucleic acidsof the tissue sample to hybridize to the poly-T capture region of thesecond probe comprises an electrophoretic transfer of the nucleic acidsfrom the tissue sample onto the capture array.
 4. The method of claim 1,wherein the first primer binding region sequence and the second primerbinding sequence are different.
 5. The method of claim 1, where in thefirst probe further comprises a universal adaptor oligonucleotide; andwherein the (e) comprises linking the immobilized first complementarystrand to the spatial address sequence of the first probe to immobilizethe first complementary strand at both ends via the universal adapteroligonucleotide.
 6. The method of claim 1, wherein the linking compriseslinking a 3′ end of the complementary strand to a 5′ end of the firstcapture probe.
 7. The method of claim 1, wherein the first complementarystrand comprises a cDNA molecule.